Composition and/or combination for aquaculture

ABSTRACT

Disclosed embodiments concern a composition and/or combination, and a method of administering the same as a feed, or to supplement the feed of, aquatic animals, particularly for aquaculture. Disclosed composition and/or combination embodiments may comprise glucan, silica, mineral clay, mannans,  yucca, quillaja , a probiotic, and/or an adhesive agent. The adhesive agent may be selected particularly to facilitate administration to aquatic species. In certain embodiments the adhesive agent comprises an oil, such as soy oil, or a syrup, such as molasses, or combinations thereof. In some embodiments the composition and/or combination may further comprise polyphenol, an antimicrobial, and/or a vaccine. Also disclosed is a method for promoting growth and/or immune function in aquatic animals.

CROSS REFERENCE TO RELATED APPLICATION

This is a U.S. continuation-in-part of International Application No.PCT/US2016/051080, filed on Sep. 9, 2016, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of the earlierfiling dates of U.S. provisional patent applications Nos. 62/216,153 and62/216,162, both filed on Sep. 9, 2015, each of which are incorporatedherein by reference in their entirety.

FIELD

The present disclosure concerns embodiments of a composition and/orcombination for use alone or in combination with a feedstuff, and amethod for administering such embodiments to aquatic animals foraquaculture.

BACKGROUND

Aquaculture is the process of raising aquatic animals, such as fish,crustaceans, and mollusks, typically but not necessarily as feed animalsfor human consumption. Aquaculture involves feeding the animals,protecting them from predators and illnesses, and generally raising theanimals in order to enhance their production and growth. In order foraquaculture to be profitable, it must be a more efficient way ofprocuring aquatic animals than harvesting wild aquatic animals.

In aquaculture, feed conversion ratios are used to provide animalproducers with a method for monitoring the efficiency of raisinganimals. The ability to estimate the amount of feed required per unit ofbody gain for animals allows animal producers to effectively budgetcosts associated with raising animals, such as feed shortfalls or waste,which can facilitate determining profit margins. Lowering the feedconversion ratio, meaning less food is required per unit of body gain,is an effective way to reduce aquaculture costs. In 1980, a report byRobert Smith for the United States Fish and Wildlife Service providedresults from trials concerned with “the effect of several types of clayon growth rate, feed conversion efficiency and mortality of rainbowtrout. Smith R. R., Recent advances in nutrition: clay in trout diets;Salmonid, 1980, vol. 4(4), 16-18. The report states that the “inclusionof some types of clay at about 10% of the diet can significantly reducefeed/grain ratios and increase rate of gain.” The clays that resulted inthese benefits were a sodium bentonite clay and a “clay supplied by‘Ion-Min’.” Ion-Min® is a brand name for montmorillonite clay productsfrom California Earth minerals.

Fish disease is a substantial source of monetary loss to aquaculturists.Production costs are increased by fish disease outbreaks because of theinvestment lost in dead fish, treatment costs, and decreased growthduring convalescence. In nature diseased fish are quickly removed fromthe population by predators. In addition, fish are much less crowded innatural systems than in captivity. Parasites and bacteria may be ofminimal significance under natural conditions, but can be substantiallyproblematic when animals are crowded and stressed under cultureconditions.

Disease is rarely a simple association between a pathogen and a hostfish. Usually other circumstances must be present for active disease todevelop in a population. These circumstances are generally circumstancesthat cause the aquatic animal “stress.”

There are two broad categories of disease that affect fish, infectiousand non-infectious diseases. Infectious diseases are caused bypathogenic organisms present in the environment or carried by otherfish. They are contagious, and some type of treatment may be necessaryto control the disease outbreak. In contrast, non-infectious diseasestypically are caused by environmental issues, nutritional deficiencies,or genetic anomalies; they are not contagious and usually cannot becured by medications.

Infectious diseases are broadly categorized as parasitic, bacterial,viral, or fungal diseases. Parasitic fish diseases are most frequentlycaused by small microscopic organisms called protozoa which live in theaquatic environment. There are a variety of protozoans that infest thegills and skin of fish causing irritation, weight loss, and eventuallydeath. Most protozoan infections are relatively easy to control usingstandard fishery chemicals such as copper sulfate, formalin, orpotassium permanganate.

Bacterial diseases are often internal infections and require treatmentwith medicated feeds containing antibiotics. Typically, fish infectedwith a bacterial disease will have hemorrhagic spots or ulcers along thebody wall and around the eyes and mouth. They may also have an enlarged,fluid-filled abdomen, and protruding eyes. Bacterial diseases can alsobe external, resulting in erosion of skin and ulceration. Columnaris isan example of an external bacterial infection which may be caused byrough handling.

Viral diseases are impossible to distinguish from bacterial diseaseswithout special laboratory tests. They are difficult to diagnose andthere are no specific medications available to cure viral infections offish.

Fungal diseases are the fourth type of infectious disease. Fungal sporesare common in the aquatic environment, but are not normally a problem inhealthy fish. When fish are infected with an external parasite,bacterial infection, or injured by handling, fungi can colonize diseasedtissue on the exterior of the fish. These areas appear to have a cottonygrowth or may appear as brown matted areas when the fish are removedfrom the water.

Non-infectious diseases can be broadly categorized as environmental,nutritional, or genetic. Environmental diseases are the most importantin commercial aquaculture. Environmental diseases include low dissolvedoxygen, high ammonia, high nitrite or natural or man-made toxins in theaquatic environment. Ammonia toxicity is one factor associated withreduced health and loss of aquatic animals in aquaculture facilities.Fish and amphibians lack the mechanisms used by humans and other mammalsto remove ammonia solutions from their bloodstream, as they can usuallyexcrete it directly. For this reason, ammonia is toxic to aquaticanimals. Excess ammonia can result in poor growth and feed conversionrates, reduced reproductive capability, and increased stress, makinganimals more susceptible to disease and infection. In fish, higherconcentrations of ammonia can also damage gills and tissue, andultimately result in death.

Nutritional diseases can be very difficult to diagnose. A classicexample of a nutritional catfish disease is “broken back disease,”caused by vitamin C deficiency. The lack of dietary vitamin Ccontributes to improper bone development, resulting in deformation ofthe spinal column. Another important nutritional disease of catfish is“no blood disease,” which may be related to a folic acid deficiency.Affected fish become anemic and may die. The condition seems todisappear when the deficient feed is discarded and a new feed provided.

Coccidiosis is a parasitic disease of the intestinal tract of animalscaused by coccidian protozoa of the genus Eimeria. The disease canspread amongst animals by contact with infected feces by means of aninfective form called the oocyst. Coccidiosis is a significant diseaseof certain animals, as it can affect animals at a very young age. It canbe fatal or compromise animal health, thereby impairing the feedconversion rate of the animals. Thus, production, reproductiveefficiency and animal health are adversely affected by coccidiosis.Diseases, such as coccidiosis, cause significant economic losses incertain animal industries. Such diseases also can negatively affect thehealth of domesticated animals.

SUMMARY

Disclosed embodiments concern a composition and/or combinationcomprising glucan, silica, mineral clay, mannans, polyphenol, yucca,quillaja, a probiotic, a combination thereof, or various combinationsthereof that can be administered separately, but potentially withinoverlapping therapeutic effective periods, and a method of administeringthe same to an aquatic animal. The composition and/or combination may beformulated for use in aquaculture, such as a formulation that issuitable to be ingested and/or eaten when administered to an aquaticspecies. The composition and/or combination may further comprise anantimicrobial, a vaccine, or a combination thereof. The antimicrobialmay be an antibiotic, an antifungal, an antiparasitic, an antiviral, ora combination thereof. In some embodiments, the antiparasitic is ananticoccidal, such as Salinomycin, and/or the antibiotic isvirginiamycin. The composition and/or combination may also compriseendoglucanohydrolase. In some embodiments, the composition and/orcombination comprises between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, and between 1% and 8.0%mannans. In a particular embodiment, the composition and/or combinationconsists essentially of β-glucans, β-1,3 (4)-endoglucanohydrolase,diatomaceous earth, a mineral clay, and glucomannan. In any embodiment,the compositions and/or combinations can also include an adhesive agentselected particularly to facilitate administration to aquatic species.In certain embodiments the adhesive agent comprises an oil, such as soyoil, or a syrup, such as molasses.

The composition and/or combination may further comprise a feedstuff. Thefeedstuff may be a feed ration, a mineral supplement, a proteinsupplement, a premix, molasses, a liquid feed, water, or any combinationthereof. The composition and/or combination is formulated as a powder, agranule, a pellet, a solution, a suspension, or a combination thereof.In some embodiments, the composition and/or combination is formulated asa solid material having a particle size of from greater than zero to 4mm or more, such as from greater than zero to 1 mm, from 1 mm to 2 mm orfrom 2 mm to 3 mm or more.

The composition and/or combination may be a composition. In otherembodiments, the composition and/or combination is a combinationcomprising two or more components formulated for substantiallysimultaneous administration, or alternatively, formulated for sequentialadministration in any order. In still further embodiments, thecombination comprises a single component administered multiple timeswith at least two administrations overlapping in an effective timeperiod, and the single component may be formulated differently foradministration at two different times.

In some embodiments, the aquatic animals can be animals raised for humanconsumption. In other embodiments the aquatic animals can be ornamentalanimals. Exemplary aquatic animals include fish (for example tilapia,carp, sea bream, or salmon), crustaceans (for example crabs, lobsters,or shrimp), and mollusks (for example octopus, oysters, or clams). Thedisclosed composition and/or combination may be administered to promotegrowth, to reduce its feed conversion ratio, to ameliorate at least onedeleterious symptom or sign, to prevent or delay the onset of at leastone deleterious symptom or sign, when the animal is exposed to astressor, or will be exposed to a stressor, such as to ameliorate theeffect of the stressor, or a combination thereof. In some embodiments,the composition and/or combination is administered to treat or preventammonia toxicity.

In any or all of the above embodiments, an effective amount of thecomposition and/or combination is administered to the aquatic species.In certain embodiments, the effective amount ranges from about 1 mg/kgbody weight per day to about 20 g/kg body weight per day. In any or allof the above embodiments, the composition and/or combination may beadministered by mixing the composition and/or combination with theanimal's feed in an amount ranging from about 0.1 to about 20 kg per tonof feed and providing the composition and/or combination mixed with thefeed to the animal. The composition and/or combination may be adhered tothe feed, such as feed pellets, granules and/or powder, by an adhesiveagent, and in some embodiments, the composition and/or combination maybe top-coated onto the feed using the adhesive agent. In someembodiments, the composition and/or combination is formulated as apowder, a granule, a pellet, a solution, or a suspension.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table illustrating exemplary dose ranges of disclosedexemplary embodiments of the composition and/or combination for variousgrowth stages.

FIG. 2 is a graph of the growth rate of sea bream using two differentdoses of exemplary embodiments of the composition and/or combination anda control group

FIG. 3 is a table comparing the average weight (g) of sea bream beingfed two different doses of exemplary embodiments of the compositionand/or combination and a control group

FIG. 4 is a table concerning a statistical analysis (ANOVA-singlefactor) of the average weight of sea bream after 158 days.

FIG. 5 is a table showing the feed conversion rate of sea bream in thetrial at day 158.

FIG. 6 is a table showing the water quality data for the sea bream trialof FIG. 2.

FIG. 7 is a graph illustrating the water temperature during the seabream trial of FIG. 2.

FIG. 8 is a table of the expected growth rate and feeding chart of seabream.

FIG. 9 is a table of growth rate results by tank during the sea breamtrial of FIG. 2.

FIGS. 10A-10J are tables concerning a statistical single-factor ANOVAanalysis for trial groups A, B, and C for each date of measure.

FIG. 11 is a table of the feed conversion ratio (FCR) by tank bytreatment.

FIGS. 12A and 12B are tables showing survival rate in the trial at day128, and the mortality in each tank by date.

FIGS. 13A-13E are photographs of the experimental set up for the seabream trial in FIG. 2.

FIG. 14 is a graph of weight verses days comparing the growth rate ofhybrid tilapia using two different composition feed groups and a controlgroup.

FIG. 15 is a table comparing the average weight (g) of hybrid tilapiabeing fed two different doses of the composition and/or combination anda control group.

FIG. 16 is a statistical single-factor ANOVA analysis of the averageweight of the hybrid tilapia after 149 days.

FIG. 17 is a table of the food conversion ratio results for each groupfrom the hybrid tilapia trial of FIG. 14.

FIG. 18 is a statistical, single-factor ANOVA analysis of the hybridtilapia food conversion ratio of FIG. 17 after 149 days.

FIG. 19 is a table showing the water quality data collected during thehybrid tilapia trial of FIG. 14.

FIG. 20 is a graph showing the water temperature versus days during thehybrid tilapia trial of FIG. 14.

FIG. 21 is a table showing the expected growth rate and feeding chart oftilapia.

FIG. 22 is a table showing the results of growth rate by cage and dateduring the hybrid tilapia trial of FIG. 14.

FIGS. 23A-23I are tables showing a statistical, single-factor ANOVAanalysis for trial groups A, B, and C for hybrid tilapia for each dateof measure during the hybrid tilapia trial of FIG. 14.

FIG. 24 is a table of the feed conversion rate values per cage andtreatment for the hybrid tilapia trial of FIG. 14.

FIGS. 25A-25C are photographs of the experimental set-up for the hybridtilapia trial of FIG. 14.

FIG. 26 is a graph of weight (g) versus days, illustrating the growthrate of common carp using two feed groups with different doses of thecomposition and/or combination and a control group.

FIG. 27 is a table comparing the average weight (g) of common carp beingfed two different doses of the composition and/or combination and acontrol group.

FIG. 28 is a statistical, single-factor ANOVA analysis of the averageweight of the common carp during the common carp trial of FIG. 26 after83 days.

FIG. 29 is a table of the food conversion ratio results for each groupduring the common carp trial of FIG. 26.

FIG. 30 is a statistical, single-factor ANOVA analysis of the foodconversion ratio of the common carp during the common carp trial of FIG.26 after 83 days.

FIG. 31 is a table providing the water quality data collected during thecommon carp trial of FIG. 26.

FIG. 32 is a graph providing the water temperature during the commoncarp trial of FIG. 26.

FIG. 33 is a table providing the feeding chart of carp by temperatureduring the common carp trial of FIG. 26.

FIG. 34 is a table providing the growth rate results by cage and dateduring the common carp trial of FIG. 26.

FIGS. 35A-35E are tables providing a statistical, single-factor ANOVAanalysis for trial groups A, B, and C for common carp for each date ofmeasure.

FIG. 36 is a table of the feed conversion rate values per cage andtreatment for the common carp trial of FIG. 26.

FIGS. 37A-37C are photographs of the experimental set-up for the commoncarp trial of FIG. 26.

FIG. 38 is a graph of mortality versus days after stress, comparing thedaily mortality of tilapia fed with the composition and/or combinationand the control group.

FIG. 39 is a graph of mortality (%) versus tank number, comparing thetotal mortality of tilapia per tank of tilapia fed with the compositionand/or combination and the control group during the tilapia trial ofFIG. 38.

FIG. 40 is a bar graph illustrating the total mortality of tilapiaseparated by treatment during the tilapia trial of FIG. 38.

FIG. 41 is a statistical, single-factor ANOVA analysis of tilapiamortalities during the tilapia trial of FIG. 38 after 74 days.

FIG. 42 is a table providing the water quality data for the tilapiatrial of FIG. 38.

FIG. 43 is a graph of level (ppm) versus time (days), illustratingammonia and nitrite levels in the water during the tilapia trial of FIG.38.

FIG. 44 is a graph of level versus time (days), illustrating pH andoxygen (ppm) levels in the water during the tilapia trial of FIG. 38.

FIG. 45 is a graph of turbidity versus time (days) illustrating thewater turbidity during the tilapia trial of FIG. 38.

FIG. 46 is a table listing the toxicity of ammonia (NH₃) inmultiplication factors.

FIG. 47 is a table listing the concentration of unionized ammonia atvarious concentrations of total ammonia and pH.

FIG. 48 is a table providing the mortality per cage by date during thetilapia trial of FIG. 38.

FIGS. 49A-49C are photographs of exemplary tilapia from the trial ofFIG. 38.

FIGS. 50A and 50B are photographs showing the experimental set up of thetilapia trial of FIG. 38.

FIG. 51 is a table comparing the average survival (percent from 4replicates) of shrimp using 100 and 200 mg/kg bodyweight per day doses,with a control.

FIG. 52 is a table listing the water quality data from the experiment ofFIG. 51.

FIG. 53 is a photograph of the experimental setup for the experiment ofFIG. 51.

FIG. 54 is a photograph showing exemplary shrimp at the end of theexperiment of FIG. 51.

FIG. 55 is a plot of percentage shrimp survival versus treatment,illustrating the four-fold increase in shrimp survival in shrimp fed anexemplary embodiment of the disclosed composition.

FIG. 56 is a plot of yield in kg per pond versus treatment, illustratingthe increase in shrimp yield in shrimp fed an exemplary embodiment ofthe disclosed composition.

FIG. 57 is a schematic cross-sectional diagram illustrating oneembodiment of a particle that comprises the composition and/orcombination, a feedstuff and an adhesive agent.

FIG. 58 is a timeline illustrating a proposed experimental shrimp trialof one embodiment of the disclosed composition and/or combination.

FIG. 59 is a graph of average survival rate versus time, illustratingthe average survival of shrimp that are fed one embodiment of thedisclosed composition and/or combination at different inclusion rates.

FIG. 60 is a graph of average body weight versus time, illustrating theaverage body weight of shrimp that are fed one embodiment of thedisclosed composition and/or combination at different inclusion rates ondays 30 and 45 of the trial.

FIG. 61 is a graph of feed conversion ratio (FCR) at day 45,illustrating the feed conversion rate at day 45 of shrimp that are fedone embodiment of the disclosed composition and/or combination atdifferent inclusion rates compared to control shrimp.

FIG. 62 is a graph of average survival rate versus time, illustratingthe average survival of shrimp that are fed one embodiment of thedisclosed composition and/or combination at different inclusion rates atdays 0, 15 and 30.

FIG. 63 is a graph of average body weight versus time, illustrating theaverage body weight of shrimp that are fed one embodiment of thedisclosed composition and/or combination at different inclusion rates ondays 15 and 30 of the challenge trial.

FIG. 64 is a graph of feed conversion ratio (FCR) at day 30,illustrating the feed conversion rate at day 30 of shrimp that are fedone embodiment of the disclosed composition and/or combination atdifferent inclusion rates compared to control shrimp in a challengetrial.

FIG. 65 is a graph of number of bacteria versus time, illustrating thetotal number of bacteria in the intestine of shrimp that are feddifferent diets.

FIG. 66 is a graph of number of Vibrio species versus time, illustratingthe total number of Vibrio species in the shrimp that are fed differentdiets.

FIG. 67 is a graph of total hemocyte count versus treatment,illustrating the hemocyte count in shrimp administered different amountsof the composition and/or combination.

FIG. 68 is a graph of phagocytosis activity versus treatment,illustrating the phagocytosis activity count in shrimp administereddifferent amounts of the composition and/or combination.

FIG. 69 is a graph of phenoloxidase activity versus treatment,illustrating the phenoloxidase activity in shrimp administered differentamounts of the composition and/or combination.

FIG. 70 is a graph of superoxide dismutase activity versus treatment,illustrating the superoxide dismutase activity in shrimp administereddifferent amounts of the composition and/or combination.

FIG. 71 is a digital image of a portion of the hepantopancreas ofcontrol shrimps, illustrating about 30% cell necrosis.

FIG. 72 is a digital image providing an expanded view of thehepantopancreas shown in FIG. 71.

FIG. 73 is a digital image of a portion of the hepantopancreas ofshrimps administered 100 mg/kg BW of one embodiment of the disclosedcomposition and/or combination, illustrating about 25% cell necrosis.

FIG. 74 is a digital image providing an expanded view of thehepantopancreas shown in FIG. 73.

FIG. 75 is a digital image of a portion of the hepantopancreas ofshrimps administered 200 mg/kg BW of one embodiment of the disclosedcomposition and/or combination, illustrating about 25% cell necrosis.

FIG. 76 is a digital image providing an expanded view of thehepantopancreas shown in FIG. 75.

DETAILED DESCRIPTION I. Terms

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Antimicrobial: An agent that kills and/or inhibits the growth ofmicroorganisms. As used herein, antimicrobials include antibiotics,antifungals, antivirals, and antiparasitics including anticoccidials, orcombinations thereof.

Administering: Administration by any route to the subject. As usedherein, administration to aquatic species typically refers to oraladministration.

Binding agent or binder: A material or substance that is used to hold ordraw together other materials to form a cohesive unit.

Combination: A combination includes two or more components that areadministered such that the effective time period of at least onecomponent overlaps with the effective time period of effective timeperiods of all components administered overlap with each other. In anexemplary embodiment of a combination comprising four components, theeffective time period of the first component administered may overlapwith the effective time periods of the second, third and fourthcomponents, but the effective time periods of the second, third andfourth components independently may or may not overlap with one another.In another exemplary embodiment of a combination comprising fourcomponents, the effective time period of the first componentadministered overlaps with the effective time period of the secondcomponent, but not that of the third or fourth; the effective timeperiod of the second component overlaps with those of the first andthird components; and the effective time period of the fourth componentoverlaps with that of the third component only. A combination may be acomposition comprising the components, a composition comprising one ormore components and another separate component (or components) orcomposition(s) comprising the remaining component(s), or the combinationmay be two or more individual components. In some embodiments, the twoor more components may comprise the same component administered at twoor more different times, two or more different components administeredsubstantially simultaneously or sequentially in any order, or acombination thereof.

Excipient or carrier: A physiologically inert substance that is used asan additive in (or with) a combination, composition, or component asdisclosed herein. As used herein, an excipient or carrier may beincorporated within particles of a combination, composition, orcomponent, or it may be physically mixed with particles of acombination, composition, or component. An excipient or carrier can beused, for example, to dilute an active agent and/or to modify propertiesof a combination or composition. Examples of excipients and carriersinclude, but are not limited to, calcium carbonate, polyvinylpyrrolidone(PVP), tocopheryl polyethylene glycol 1000 succinate (also known asvitamin E TPGS, or TPGS), dipalmitoyl phosphatidyl choline (DPPC),trehalose, sodium bicarbonate, glycine, sodium citrate, and lactose.

Feed conversion rate: A measure of the efficiency of an animal toconvert feed mass into increased body mass; also known in the art asfeed conversion ratio (which is expressed herein as a dimensionlessnumber).

Feedstuff: Anything that may be consumed by an animal. The term“feedstuff” includes, but is not limited to, solid and liquid animalfeeds (e.g., a feed ration), supplements (e.g., a mineral supplement),water, and feed additive carriers (e.g., molasses).

Mannans: A class of polysaccharides including the sugar mannose. Themannans family includes pure mannans (i.e., the polymer backbonecomprises of mannose monomers), glucomannan (the polymer backbonecomprises mannose and glucose), and galactomannan (mannans orglucomannan in which single galactose residues are linked to the polymerbackbone). Mannans are found in cell walls of some plant species andyeasts, and may be provided as extracts of such plant species and/oryeasts.

Mineral clay: The term “mineral clay” refers to hydrous aluminumsilicates. Mineral clays usually include minor amounts of impurities,such as potassium, sodium, calcium, magnesium, and/or iron. Mineralclays typically have a two-layer sheet structure including tetrahedralsilicate sheets and octahedral hydroxide sheets or a three-layerstructure including a hydroxide sheet between two silicate sheets.

Polyphenols: A class of natural, synthetic, or semisynthetic organicchemicals characterized by the presence of plural phenolic

structural units.

Saponin: A class of chemical compounds, one of many secondarymetabolites found in natural sources, with saponins found in particularabundance in various plant species. More specifically, they areamphipathic glycosides grouped, in terms of structure, by theircomposition. In certain embodiments, saponin comprises one or morehydrophilic glycoside moieties combined with a lipophilic triterpenederivative.

Therapeutic agent: An agent that is capable of providing a therapeuticeffect, e.g., preventing a disorder, inhibiting a disorder, such as byarresting the development of the disorder or its clinical symptoms, orrelieving a disorder by causing regression of the disorder orameliorating its clinical symptoms.

Therapeutically effective amount: A quantity or concentration of aspecified compound, composition or combination sufficient to achieve aneffect in a subject.

Additional disclosure is provided by U.S. patent application Ser. No.14/699,740, U.S. patent application Ser. No. 13/566,433, U.S. patentapplication Ser. No. 13/872,935, U.S. Patent Publication No.2013/0017211, U.S. Patent Publication No. 2012/0156248, U.S. PatentPublication No. 2007/0253983, U.S. Patent Publication No. 2007/0202092,U.S. Patent Publication No. 20070238120, U.S. Patent Publication No.2006/0239992, U.S. Patent Publication No. 2005/0220846, U.S. PatentPublication No. 2005/0180964, and Australian Patent Application No.2011201420, each of which is incorporated herein by reference in itsentirety.

II. Compositions and/or Combinations

A. Compositions and/or Combinations Comprising Glucan, Silica, MineralClay and/or Mannans

Disclosed herein are embodiments of compositions and/or combinations foruse in aquaculture and formulated for feeding to aquatic animalsincluding, but not limited to, fish, crustaceans, and mollusks. Aquaticanimals may be raised for human consumption, ornamental use, or otherreasons.

Certain disclosed embodiments of the composition and/or combinationcomprise glucan (e.g., β-1,3 (4)glucan), silica, mineral clay, mannansor any combination thereof, and may further comprise an adhesive agent.In some embodiments, the composition and/or combination furthercomprises an endoglucanohydrolase, such as β-1,3(4)-endoglucanohydrolase, either endogenously or as an affirmativelyadded ingredient.

The composition and/or combination may comprise, consist essentially of,or consist of, glucan (e.g., β-1,3 (4)glucan), silica, mineral clay andmannans. In some embodiments, the composition and/or combinationcomprises, consists essentially of, or consists of, glucan (e.g., β-1,3(4)glucan), silica, mineral clay, mannans and endoglucanohydrolase. Inother embodiments, the composition and/or combination comprises,consists essentially of, or consists of, glucan (e.g., β-1,3 (4)glucan),silica, mineral clay, mannans, endoglucanohydrolase and an adhesiveagent. In further embodiments, the composition and/or combinationcomprises, consists essentially of, or consists of, glucan (e.g., β-1,3(4)glucan), silica, mineral clay, mannans, endoglucanohydrolase, anadhesive agent and a feedstuff.

The composition and/or combination may comprise, consist essentially of,or consist of, silica, mineral clay and yeast cell wall extract. In someembodiments, the composition and/or combination comprises, consistsessentially of, or consists of, silica, mineral clay, yeast cell wallextract and an adhesive agent. In other embodiments, the compositionand/or combination comprises, consists essentially of, or consists of,silica, mineral clay, yeast cell wall extract, an adhesive agent and afeedstuff.

The composition and/or combination may comprise glucan, silica, mineralclay or mannans. Alternatively, the composition and/or combination maycomprise glucan and silica, glucan and mineral clay, glucan and mannans,silica and mineral clay, silica and mannans, or mineral clay andmannans. In some embodiments, the composition and/or combinationcomprises glucan, silica and mineral clay, glucan, silica and mannans,or silica, mineral clay and mannans, and in certain embodiments, thecomposition and/or combination comprises glucan, silica, mineral clayand mannans. Any of these embodiments may further comprise an adhesiveagent. Exemplary such embodiments include, but are not limited to,silica, glucan, and adhesive agent; glucan, mineral clay, mannans, andadhesive agent; glucan, silica, mineral clay, mannans, and an adhesiveagent; or silica, mineral clay, yeast cell wall extract and an adhesiveagent. Additionally, or alternatively, any of these embodiments mayfurther comprise an endoglucanohydrolase.

In some embodiments, the components are in a composition. In otherembodiments, the components are in a combination, which may compriseindividual components, or may comprise one or more compositions eachcomprising one or more components, and/or other separate components.

Suitable sources of silica include, but are not limited to, sand,diatomaceous earth, and synthetic silica. In one embodiment, quartz maybe used. In certain embodiments, the mannans comprise glucomannan.

The components of the composition and/or combination are prepared bymethods commonly known in the art and can be obtained from commercialsources. β-1,3 (4)-endoglucanohydrolase may be produced from submergedfermentation of a strain of Trichoderma longibrachiatum. Diatomaceousearth is available as a commercially-available, acid-washed product with95% silica (SiO₂) and with its remaining components not assayed butprimarily ash (minerals) as defined by the Association of AnalyticalChemists (AOAC, 2002). The mineral clays (e.g., aluminosilicates) usedin this composition and/or combination may be any of a variety ofcommercially-available clays including, but not limited to,montmorillonite clay, bentonite and zeolite. Glucan, mannans, and/orendoglucanohydrolase can be obtained from plant cell walls, yeast (e.g.,Saccharomyces cerevisiae, Candida utilis), certain fungi (e.g.,mushrooms), and bacteria. In certain embodiments, yeast can beaffirmatively administered to provide glucan, mannans andendoglucanohydrolase endrogeneously.

In one embodiment, the composition and/or combination includes 1-40 wt %silica, 1-25 wt % glucan and mannans, and 40-92 wt % mineral clay inamounts relative to each other. In another embodiment, the compositionand/or combination comprises 5-40 wt % silica, 2-15 wt % glucan andmannans, 40-80 wt % mineral clay in amounts relative to each other,and/or an effective amount of adhesive agent, such as at least 2 wt %adhesive agent. In another embodiment, the composition and/orcombination comprises 20-40 wt % silica, 4-10 wt % glucan and mannans,50-70 wt % mineral clay in amounts relative to each other, and/or aneffective amount of an adhesive agent, such as at least 2 wt % adhesiveagent. In another embodiment, the composition and/or combinationcomprises 15-40 wt % silica, 1-15 wt % glucans, 0-10 wt % mannans, 50-81wt % mineral clay in amounts relative to each other, and/or an effectiveamount of adhesive agent, such as at least 2% adhesive agent. In anotherembodiment, the composition and/or combination comprises 15-40 wt %silica, 1.0-5.0 wt % glucans, 1.0-8.0 wt % mannans, 50-81 wt % mineralclay in amounts relative to each other, and/or an effective amount ofadhesive agent, such as at least 2 wt % adhesive agent. In anotherembodiment, the composition and/or combination comprises 20-30 wt %silica, 1.0-3.5 wt % glucans, 1.0-6.0 wt % mannans, 60-75 wt % mineralclay in amounts relative to each other, and/or an effective amount ofadhesive agent, such as at least 2% adhesive agent.

In some embodiments, β-glucans and mannans are obtained from yeast oryeast cell wall extract. The composition and/or combination may comprise1-40 wt % silica, 1-30 wt % yeast and 40-92 wt % mineral clay, or 1-40wt % silica, 1-30 wt % yeast cell wall extract, 40-92 wt % mineral clay,in amounts relative to each other. In one embodiment, the compositionand/or combination comprises 10-40 wt % silica, 5-20 wt % yeast cellwall extract, 40-80 wt % mineral clay in amounts relative to each other,and/or at least 2% adhesive agent. In another embodiment, thecomposition and/or combination comprises 15-30 wt % silica, 5-15 wt %yeast cell wall extract, 55-70 wt % mineral clay in amounts relative toeach other, and/or at least 2% adhesive agent.

In any of the above embodiments, the composition and/or combination mayfurther comprise an endoglucanohydrolase, such as β-1,3(4)-endoglucanohydrolase. The composition and/or combination may includefrom 0.05 wt % endoglucanohydrolase to 5 wt % endoglucanohydrolase ormore, such as from 0.05 wt % to 3 wt % β-1,3 (4)-endoglucanohydrolase,relative to the amounts of silica, mineral clay, glucan, mannans, and/oryeast cell wall present in the composition and/or combination. In oneembodiment, the composition and/or combination consists essentially of0.1-3 wt % β-1,3 (4)-endoglucanohydrolase, 20-40 wt % silica, 2-20 wt %glucan and mannans, 50-70 wt % mineral clay, and/or at least 2% adhesiveagent. In another embodiment, the composition and/or combinationconsists essentially of 0.2-3 wt %, β-1,3 (4)-endoglucanohydrolase,20-40 wt % silica, 4-10 wt % glucan and mannans, 50-70 wt % mineralclay, and/or at least 2% adhesive agent. In any of the aboveembodiments, the silica may be provided by diatomaceous earth. In any ofthe above embodiments, the glucans may be β-glucans. In someembodiments, the β-glucans can be obtained from yeast, or othermaterials, such as fungi, algae, or the like. In any of the aboveembodiments, the mannans may comprise glucomannan.

The glucan and mannans (or yeast or yeast cell wall extract) can beprepared by a method known to a person of ordinary skill in the art andas further disclosed by the patent documents incorporated herein byreference. Yeast cell wall extract may have a composition comprising0-8% moisture and 92-100% dry matter. The dry matter may comprise 10-55%protein, 0-25% fats, 0-2% phosphorus, 10-30% β-glucan, 0-25% mannans,and 0-5% ash. In an independent embodiment, a commercial source of β-1,3(4) glucan and glucomannan derived from primary inactivated yeast(Saccharomyces cerevisiae) with the following chemical composition canbe used: moisture 3.5-6.5%; proteins 1-6%; fats 0-0.5%; phosphorus0-0.2%; mannans 9-20%; β-1, 3-(4) glucan 9-18%; and ash 75-85%.

In another independent embodiment, the yeast cell wall extract comprisesmoisture 2-3% and dry matter 97-98%, and the dry matter may compriseproteins 14-17%, fats 20-22%, phosphorus 1-2%, mannans 22-24%, β-1,3-(4) glucan 24-26%, and ash 3-5%.

In an independent embodiment of the composition and/or combination,silica, glucan and mannans, and mineral clay are combined at 1-40%,1-25% and 40-92%, respectively. In an independent embodiment of thecomposition and/or combination, β-1,3 (4)-endoglucanohydrolase,diatomaceous earth, yeast cell wall extract and mineral clay arecombined at 0.05-3%, 1-40%, 1-20% and 40-92%, respectively. In anindependent composition and/or combination, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cell wall extractand mineral clay are combined at 0.1-3%, 5-40%, 2-10% and 40-80%,respectively. In another independent embodiment of the compositionand/or combination, β-1,3 (4)-endoglucanohydrolase, diatomaceous earth,yeast cell wall extract and mineral clay are combined at 0.2-3%, 30-40%,4-6% and 50-65%, respectively.

B. Composition and/or Combinations Comprising Polyphenols

Additionally, or alternatively, the composition and/or combination maycomprise a polyphenol. In some embodiments, the polyphenol is in a plantextract. Embodiments of disclosed plant extracts can be prepared frompolyphenol-containing plant material. The plant material also mayinclude non-polyphenol compounds, including polyphenol degradationproducts, such as gallic acid and trans-caftaric acid. Degradation canoccur, for example, through oxidative and/or biological processes. Boththe polyphenols and the non-polyphenol compounds may have biologicalactivity. The plant extract may be prepared from a single plant material(e.g., grapes) or from a combination of plant materials. In someembodiments, the plant extract is prepared from a pressed plantmaterial, such as grape pomace, a dried plant material, such as tea, ora combination thereof. Pomace may be obtained substantially immediatelypost-pressing or as an ensiled product, i.e., pomace collected andstored for up to several months post-pressing. Suitable plants have aplurality of polyphenols and/or other non-polyphenolic compounds,including but not limited to non-polyphenolic organic acids (such asgallic acid and/or trans-caftaric acid), flavanols, gallate esters,flavanodiols, phloroglucinol, pyrogallol, and catechol. In someembodiments, the plant extract is prepared from Pinot noir pomace, Pinotgris pomace, or green tea.

In some embodiments, pressed or dried plant material is ground to a finepowder prior to, or during, extraction. Pressed plant materials may befrozen to facilitate grinding. Polyphenols and other non-polyphenoliccompounds may be extracted for administration. For example, polyphenolsand other non-polyphenolic compounds may be extracted from the powderusing a solution comprising a polar solvent, such as water, an alcohol,an ester, or a combination thereof. In some embodiments, the solutioncomprises a water-miscible alcohol, ester, or combination thereof, suchas a lower alkyl alcohol, lower alkyl ester, or a combination thereof.In some embodiments, the solution is water or an aqueous solutioncomprising 25-99% of a non-aqueous solvent, such as 25-95% non-aqueoussolvent, 30-80% non-aqueous solvent, or 50-75% non-aqueous solvent, andwater. In certain embodiments, the solution is an aqueous solutioncomprising methanol, ethanol, isopropanol, ethyl acetate, or acombination thereof. The solution may be acidified by addition of anacid. The acid may prevent or minimize oxidative degradation ofbiologically-active polyphenols and other non-polyphenolic compounds inthe extract. The acid may be any suitable acid, such as a mineral acid(e.g., hydrochloric acid, sulfuric acid, nitric acid, etc.), or anorganic acid, such as citric acid or acetic acid. In some embodiments,the solution comprises from 0.01% to 1% acid, such as 0.02-0.5%,0.025-0.25%, or 0.05-0.15%. In some examples, the solution includes 0.1%hydrochloric acid.

Extraction may be performed at a temperature ranging from 0-100° C. Insome embodiments, extraction is performed at a temperature ranging from20-70° C., or at ambient temperature. Extraction is performed for aperiod of time effective to extract a polyphenol or polyphenols, such asfor a period ranging from several minutes to several days. To increaseextraction efficiency, the plant material and solution may be mixed oragitated during extraction, such as by grinding the plant materialduring extraction, stirring the mixture, shaking the mixture, orhomogenizing the mixture. In some embodiments, the extraction may berepeated one or more times with fresh solution to increase recovery ofpolyphenols and other non-polyphenolic compounds from the plantmaterial. The liquid phases from each extraction cycle are then combinedfor further processing.

The liquid phase can be recovered, and the residual solids, or pulp,discarded. Recovering the liquid phase may comprise decanting the liquidfrom the remaining solids and/or filtering the liquid phase to removeresidual solids. The solvent (alcohol, ester, or combination thereof)can be removed from the liquid solution by any suitable means, such asevaporation (e.g., roto-evaporation), to produce an aqueous extractcontaining the biologically-active components in a mildly acidicsolution.

In certain embodiments where the plant material includes a significantamount of oils, or lipids, an initial extraction of nonpolar componentsmay be performed before extracting the polyphenols and other polar,non-polyphenolic compounds. Nonpolar components may be extracted byhomogenizing the plant material in a nonpolar solvent, e.g., hexanes,heptanes, or a combination thereof. The solvent layer including theextracted nonpolar components is separated from the plant material anddiscarded.

The aqueous plant extract may be further purified by suitable means,e.g., extraction, chromatographic methods, distillation, etc., to removenon-polyphenolic compounds and/or to increase the concentration ofpolyphenols relative to other compounds in the extract.

The aqueous plant extract may be dried, for example by freeze-drying orother low-temperature drying methods, and ground to a powder to providea dried plant extract. In some embodiments, the dried plant extractcomprises 0.01 wt % to 25 wt % total polyphenols, such as 0.01 wt % to10 wt %, 0.01 wt % to 5 wt %, 0.01 wt % to 2.5 wt %, 0.01 wt % to 1 wt%, 0.01 wt % to 0.5 wt %, 0.02 to 0.25 wt %, or 0.03-0.1 wt % totalpolyphenols. In certain embodiments, the dried plant extract furthercomprises non-polyphenolic compounds. For example, the dried plantextract may comprise 0.01-1 mg/g gallic acid, such as 0.05-0.5 mg/g or0.09-0.25 mg/g gallic acid, and/or 0.001-0.1 mg/g trans-caftaric acid,such as 0.005-0.05 mg/g or 0.01-0.025 mg/g trans-caftaric acid.

The aqueous plant extract may be concentrated to a smaller volume, e.g.,by evaporation, and used as an aqueous plant extract. In otherembodiments, the aqueous plant extract is mixed with a carrier beforedrying and grinding. Suitable carriers include, for example,diatomaceous earth, silica, maltodextrin, ground grain (e.g., corn),meals (e.g., soybean or cottonseed meal) by-products (e.g., distiller'sdried grains, rice hulls, wheat mill run), clays (e.g., bentonite), andcombination thereof. The plant extract may be combined with a carrier inany suitable ratio, such as a ratio ranging from 10:1 to 1:10 by weight,such as from 5:1 to 1:5. For example, the plant extract may be mixedwith diatomaceous earth in a ratio of 3:1 by weight.

C. Quillaja and Yucca Compositions and/or Combinations

Additionally, or alternatively, the composition and/or combination maycomprise yucca, quillaja or both. In some embodiments a probiotic, forexample Bacillus coagulans, may also be added. In some embodiments,disclosed combinations and/or compositions comprising yucca, quillajaand/or a bacillus species also can improve the feed conversion rate ofcertain animals that are raised for human consumption, for example fish,crustaceans and/or mollusks. In yet additional embodiments, thecombinations and/or compositions can be used to improve animal healthgenerally.

Examples of yucca include, but are not limited to, Yucca aloifolia,Yucca angustissima, Yucca arkansana, Yucca baccata, Yucca baileyi, Yuccabrevifolia, Yucca campestris, Yucca capensis, Yucca carnerosana, Yuccacernua, Yucca coahuilensis, Yucca constricta, Yucca decipiens, Yuccadeclinata, Yucca de-smetiana, Yucca elata, Yucca endlichiana, Yuccafaxoniana, Yucca filamentosa, Yucca filifera, Yucca flaccida, Yuccagigantean, Yucca glauca, Yucca gloriosa, Yucca grandiflora, Yuccaharrimaniae, Yucca intermedia, Yucca jaliscensis, Yucca lacandonica,Yucca linearifolia, Yucca luminosa, Yucca madrensis, Yucca mixtecana,Yucca necopina, Yucca neomexicana, Yucca pallida, Yucca periculosa,Yucca potosina, Yucca queretaroensis, Yucca reverchonii, Yucca rostrata,Yucca rupicola, Yucca schidigera, Yucca schottii, Yucca sterilis, Yuccatenuistyla, Yucca thompsoniana, Yucca treculeana, Yucca utahensis, orYucca valida. In certain disclosed working embodiments, the Yucca wasYucca schidigera.

Examples of quillaja include, but are not limited to, Quillajabrasiliensis, Quillaja lanceolata, Quillaja lancifolia, Quillajamolinae, Quillaja petiolaris, Quillaja poeppigii, Quillaja saponaria,Quillaja sellowiana, or Quillaja smegmadermos.

A person of ordinary skill in the art will appreciate that, as usedherein, a plant name may refer to the plant as a whole, or to any partof the plant, such as the roots, stem or trunk, bark, leaves, flower,flower stems, or seeds or a combination thereof. These plant parts maybe used fresh, or dried, and may be whole, pulverized, or comminuted.The name may also refer to extracts from any part or parts of the plant,such as chemical extracts, or extracts obtained by pressing, or anyother methods of concentrating or extracting oils or other extractsknown to those in the art or that are hereafter discovered. Plantextracts may include compounds that are saponins, triterpenoids,polyphenols, antioxidants or resveratrol, or combinations thereof.

A composition comprising yucca and/or quillaja may also include carriersand binding agents suitable to formulate the yucca and/or quillaja foradministration to an animal. In certain working embodiments, thecomposition can be a commercially available product, such as acomposition comprising Yucca schidigera and Quillaja saponaria, which issold under the trade name NUTRAFITO PLUS by Desert King Internationaland/or MAGNI-PHI by Phibro Animal Health Corporation. Such compositionembodiments can comprise 85% Quillaja saponaria and 15% Yucca schidigeraor 90% Quillaja saponaria and 10% Yucca schidigera.

The combination and/or composition may also comprise a probiotic. Theprobiotic may be Bacillus. Bacillus is a genus of Gram-positive,rod-shaped bacteria. Examples of Bacillus include, but are not limitedto B. alcalophilus, B. alvei, B. aminovorans, B. amyloliquefaciens, B.aneurinolyticus, B. anthracis, B. aquaemaris, B. atrophaeus, B.boroniphilus, B. brevis, B. caldolyticus, B. centrosporus, B. cereus, B.circulans, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B.galliciensis, B. globigii, B. infernus, B. larvae, B. laterosporus, B.lentus, B. licheniformis, B. megaterium, B. mesentericus, B.mucilaginosus, B. mycoides, B. natto, B. pantothenticus, B. polymyxa, B.pseudoanthracis, B. pumilus, B. schlegelii, B. sphaericus, B.sporothermodurans, B. stearothermophilus, B. subtilis, B.thermoglucosidasius, B. thuringiensis, B. vulgatis, B.weihenstephanensis or a combination thereof. In some embodiments, theprobiotic is, or comprises Bacillus coagulans. In some embodiments, theprobiotic is, or comprises Bacillus subtillus. In some embodiments, theprobiotic is, or comprises Bacillus amyloliquefaciens. In someembodiments, the probiotic is, or comprises Bacillus licheniformis. Incertain embodiments, the probiotic is, or comprises, a combination ofBacillus subtillus, Bacillus amyloliquefaciens, and Bacilluslicheniformis. In other embodiments, the probiotic is or comprisesBacillus subtillus, Bacillus amyloliquefaciens, Bacillus licheniformisand Bacillus coagulans. In particular disclosed embodiments the Bacillusis Bacillus coagulans. A person of ordinary skill in the art willappreciate that, as used herein, the bacterial name may refer to thebacteria, or to a compound or compounds obtained from that bacteria.Methods of obtaining compounds from bacteria are well known in the art.

A composition comprising bacillus may also include additional materials,such as carriers or binding agents, suitable to formulate the Bacillusfor administration to an animal. In certain disclosed workingembodiments, a composition comprising Bacillus coagulans was Ganpro®, acommercial product available from Ganeden Biotech, Ohio. In otherdisclosed working embodiments, a composition comprising Bacilluscoagulans was Provia 6086®, available from Prince Agri Products, Inc.

D. Therapeutic Compositions and/or Combinations

In some embodiments, the disclosed composition and/or combinationembodiments may be administered prophylactically to an animal to reducethe risk of the animal developing particular diseases.

The compositions and/or combinations may comprise the glucan, silica,mineral clay, and mannans compositions and/or combinations describedherein, and/or may comprise the yucca and quillaja compositionsdescribed above. The composition and/or combination may further comprisean antimicrobial such as an antiparasitic (for example, ananticoccidial), an antifungal, an antibiotic, an antiviral agent, or acombination thereof; a vaccine, for example a coccidiosis vaccine; orsome combination thereof. The composition and/or combination componentsmay be administered in any order. In some embodiments, an antimicrobial,and/or a vaccine may be administered to the animal prior toadministration of glucan, mannans, mineral clay, silica, polyphenol,yucca, quillaja, probiotic or combination thereof. Alternatively, anantimicrobial and/or vaccine can be administered to an animal, followedby administration of the glucan, mannans, mineral clay, silica,polyphenol, yucca, quillaja, probiotic or combination thereof. In suchembodiments, the antimicrobial and/or vaccine may be administeredsimultaneously with any or all of the glucan, mannans, mineral clay,silica, polyphenol, yucca, quillaja, probiotic or combination thereof,or before or after any or all of the components are administered. In anindependent embodiment, an antimicrobial need not be administered. Inyet other independent embodiments, a vaccine need not be administered.

An antimicrobial may be selected from an antibiotic, an antifungal, anantiparasitic, an antiviral, or a combination thereof. An antibiotic maybe selected from, by way of example, and without limitation,virginiamycin, Bacitracin MD, Zinc Bacitracin, Tylosin, Lincomycin,Flavomycin, bambermycins, Terramycin, Neo-Terramycin, florfenicol,oxolinic acid, oxytetracycline, hydrogen peroxide (Perox-Aid® 35%),bronopol (2-bromo-2-nitro-1,3-propanediol, Pyceze®), sulfadimethozine,ormetoprim, Sulfadiazine, Trimethoprim, or a combination thereof. Insome embodiments, the antibiotic is not, or does not comprise, hydrogenperoxide. In some embodiments, the antibiotic is virginiamycin,Bacitracin MD, Zinc Bacitracin, Tylosin, Lincomycin, Flavomycin,bambermycins, Terramycin, Neo-Terramycin, florfenicol, oxolinic acid,oxytetracycline, bronopol (2-bromo-2-nitro-1,3-propanediol, Pyceze®),sulfadimethozine, ormetoprim, Sulfadiazine, Trimethoprim, or acombination thereof.

An antifungal may be selected from, by way of example, formalin,formalin-F, bronopol (2-bromo-2-nitro-1,3-propanediol, Pyceze®), or acombination thereof. Exemplary antiparasitics may be selected from ananticoccidal, copper sulfate, fenbendazole, formalin, formalin-F,hyposalinity, hadaclean A, praziquantel, emamectin benzoate (SLICE®), ora combination thereof.

Suitable anticoccidial agents include, but are not limited to,ionophores and chemical anticoccidial products. Ionophores can include,but are not limited to, Monensin, Salinomycin, Lasalocid, Narasin,Maduramicin, Semduramicin, or combinations thereof.

Chemical anticoccidial products can include, but are not limited to,Nicarbazin, Maxiban, Diclazuril, Toltrazuril, Robenidine, Stenorol,Clopidol, Decoquinate, DOT (zoalene), Amprolium, or combinationsthereof.

Suitable vaccines can be selected from live coccidiosis vaccines, suchas COCCIVAC (e.g., a composition comprising live oocysts of Eimeriaacervulina, Eimeria mivati, Eimeria maxima, Eimeria mitis, Eimeriatenella, Eimeria necatrix, Eimeria praecox, Eimeria brunetti, Eimeriahagani, or combinations thereof), LivaCox (a composition comprising300-500 live sporulated oocysts of each attenuated line of Eimeriaacervulina, E. maxima and E. tenella in a 1% w/v aqueous solution ofChloramine B), ParaCox (a composition comprising live sporulated oocystsderived from E. acervulina HP, E. brunetti HP, E. maxima CP, E. maximaMFP, E mitis HP, E. necatrix HP, E. praecox HP, E. tenella HP, andcombinations thereof), Hatch Pack Cocci III (a composition comprisingoocysts derived from Eimeria acervulina, Eimeria maxima, Eimeriatenella, or combinations thereof), INOVOCOX (a composition comprisingoocysts derived from Eimeria acervulina, Eimeria maxima, Eimeriatenella, and a sodium chloride solution), IMMUCOX (a compositioncomprising live oocysts derived from Eimeria acervulina, Eimeria maxima,Eimeria necatrix, Eimeria tenella, and combinations thereof), Advent, orcombinations thereof. Vaccines may also comprise live oocysts of theEimeria genus, for example, Eimeria aurati, Eimeria baueri, Eimerialepidosirenis, Eimeria leucisci, Eimeria rutile, Eimeria carpelli,Eimeria subepithelialis, Eimeria funduli and/or Eimeria vanasi. Vaccinesmay also comprise oocysts from the genus Epeimeria, a new genus ofcoccidia infecting fishes.

Other suitable vaccines include, but are not limited to, ALPHA DIP®2000, ALPHA DIP® Vibrio, ALPHA MARINE® Vibrio, ALPHA DIP® ERM Salar,ALPHA JECT Micro® 1 ILA, ALPHA JECT Micro® 7ILA, ALPHA JECT® Panga,ALPHA JECT® 1000, ALHPA JECT® 2000, ALPHA JECT® 3000, ALPHA JECT® 3-3,ALPHA JECT® 4000, ALPHA JECT® 4-1, ALPHA JECT® 5-1, ALPHA JECT® 5-3,ALPHA JECT® 6-2, ALPHA JECT® micro 1 ISA, ALPHA JECT® micro 2, ALPHAJECT® micro 4, Apex®-IHN, AQUAVAC® ERM Oral, AQUAVAC® ERM immersion,AQUAVAC® FNM Injectable, AQUAVAC® IPN Oral, AQUAVAC® RELERA™, AQUAVAC®Vibrio Oral, AQUAVAC® Vibrio Pasteurella injection, AQUAVAC® Vibrioimmersion and injectable, AQUAVAC-COL™ immersion, AQUAVAC-ESC™immersion, Birnagen Forte 2, Ermogen, Forte Micro, Forte V II, Forte V1,Fry Vacc 1, Furogen Dip, ICTHIOVAC JG injection, ICTHIOVAC® PDimmersion, Lipogen DUO, Lipogen Forte, Microvib, Norvax® Compact PDinjection, Norvax® Minova 4WD, Norvax® Minova 6 injection, Norvax® STREPSi immersion and injection, Premium Forte Plus, Premium Forte Plus ILA,Renogen, Vibrogen 2, or a combination thereof.

The amount of antimicrobial used is within the amounts stated below butmay depend on the particular antimicrobial used as will be understood bya person of ordinary skill in the art. In some embodiments, the amountof the antimicrobial that is included in the composition and/orcombination can range from at least 1 g/ton of feed to 230 g/ton of feed(or at least 1.1 ppm to 256 ppm), such as at least 1 g/ton of feed to220 g/ton of feed (or at least 1.1 ppm to 243 ppm), at least 1 g/ton offeed to 100 g/ton of feed (or at least 1.1 ppm to 110 ppm), at least 1g/ton of feed to 50 g/ton of feed (or at least 1.1 ppm to 55 ppm), or atleast 1 g/ton of feed to 10 g/ton of feed (or at least 1.1 ppm to 11ppm). Particular antimicrobials that can be used, and dosage amounts ofsuch antimicrobials include, but are not limited to, the following:Virginiamycin in an amount ranging from 5 g/ton of feed to 25 g/ton offeed (or 5 ppm to 27 ppm, such as 22 ppm); Bacitracin MD in an amountranging from 40 g/ton of feed to 220 g/ton of feed (or 44 ppm to 242ppm, or 50 ppm to 250 ppm in some other embodiments); Zinc Bacitracin inan amount ranging from 40 g/ton of feed to 220 g/ton of feed (or 44 ppmto 242 ppm); Tylosin in an amount ranging from 1 g/ton of feed to 1000g/ton of feed (or 1 ppm to 1100 ppm); Lincomycin in an amount rangingfrom 1 g/ton of feed to 5 g/ton of feed (or 1 ppm to 6 ppm); Flavomycinin an amount ranging from 1 g/ton of feed to 5 g/ton of feed (or 1 ppmto 6 ppm); or combinations thereof.

The amount of anticoccidial agent, as will be understood by a person ofordinary skill in the art (e.g., a veterinarian), can be selecteddepending on the particular anticoccidial agent used. In someembodiments, the amount of the anticoccidial agent that is included inthe composition and/or combination can range from at least 1 g/ton offeed to 250 g/ton of feed (or at least 1 ppm to 275 ppm), such as atleast 1 g/ton of feed to 200 g/ton of feed (or at least 1 ppm to 242ppm), or at least 1 g/ton of feed to 150 g/ton of feed (or at least 1ppm to 165 ppm), at least 1 g/ton of feed to 100 g/ton of feed (or atleast 1 ppm to 110 ppm), or at least 1 g/ton of feed to 50 g/ton of feed(or at least 1 ppm to 55 ppm). Particular anticoccidial agents that canbe used, and dosage amounts of such anticoccidial agents include, butare not limited to, the following: Monensin in an amount ranging from 35g/ton of feed to 110 g/ton of feed (or 38 ppm to 121 ppm); Salinomycinin an amount ranging from 25 g/ton of feed to 90 g/ton of feed (or 27ppm to 99 ppm); Lasalocid in an amount ranging from 35 g/ton of feed to113 g/ton of feed (or 38 ppm to 125 ppm); Narasin in an amount rangingfrom 35 g/ton of feed to 72 g/ton of feed (or 38 ppm to 79 ppm);Maduramicin in amount ranging from 2 g/ton of feed to 7 g/ton of feed(or 2 ppm to 8 ppm); Semduramicin in an amount ranging from 12 g/ton offeed to 23 g/ton of feed (or 13 ppm to 25 ppm); Nicarbazin in an amountranging from 60 g/ton of feed to 113 g/ton of feed (or 66 ppm to 125ppm); Maxiban in an amount ranging from 40 g/ton of feed to 90 g/ton offeed (or 44 ppm to 99 ppm); Diclazuril in an amount ranging from 0.5g/ton of feed to 10 g/ton of feed (or 0.6 ppm to 11 ppm); Toltrazuril inan amount ranging from 1 g/ton of feed to 10 g/ton of feed (or 1 ppm to11 ppm); Robenidine in an amount ranging from 20 g/ton of feed to 60g/ton of feed (or 22 ppm to 66 ppm); Stenorol in an amount ranging from1.5 g/ton of feed to 15 g/ton of feed (or 1.5 ppm to 17 ppm); Clopidolin an amount ranging from 90 g/ton of feed to 227 g/ton of feed (or 99ppm to 250 ppm); Decoquinate in an amount ranging from 18 g/ton of feedto 27 g/ton of feed (or 19 ppm to 29 ppm); Zoalene in an amount rangingfrom 25 g/ton of feed to 113 g/ton of feed (or 28 ppm to 125 ppm);Amprolium in an amount ranging from 20 g/ton of feed to 227 g/ton offeed (or 22 ppm to 250 ppm).

E. Miscellaneous Additives

In some embodiments the composition and/or combination further comprisesa vitamin, a trace mineral, a bulking agent, a carrier, a colorant, ataste enhancer, or any combination thereof. In other embodiments thecombination and/or composition further comprises corn, soybean meal,wheat, barley, rye, canola, corn oil, limestone, salt, distillers driedgrains with solubles (DDGS), dicalcium phosphate, sodiumsesquicarbonate, methionine source, lysine source, L-threonine, choline,or any combination thereof.

F. Adhesive Agent

In some embodiments, the composition and/or combination includes anadhesive agent. The amount of adhesive agent may be from zero to 10% ormore by weight, such as from greater than zero to 10% or from 2% to 10%by weight. The adhesive agent is a material selected to, for example,facilitate adhering some or all of the components of the compositionand/or combination together, to a foodstuff, or both. The adhesive agentalso may facilitate maintaining adherence of the composition and/orcombination together or to a foodstuff in an aquatic environment tofacilitate administration to aquatic species. The material is alsopreferably palatable and edible by aquatic animals.

In some embodiments the adhesive agent is an oil. For example, the oilmay be selected from corn oil, coconut oil, linseed oil cottonseed oil,olive oil, peanut oil, palm oil, canola oil, safflower oil, soy oil,sunflower oil, Naskole oil, or any combination thereof. In someembodiments, the adhesive agent is a syrup. For example, the syrup maybe selected from molasses, sorghum, sugar syrup, honey, or anycombination thereof. Combinations of oils and syrups also may be used.

G. Feed

The composition and/or combination may be used to replace or supplementanimal feedstuffs. In some embodiments, the feedstuff is a commercialfeedstuff. In particular embodiments, the feedstuff was manufactured byRaanan Fish Meal. The feed may be formulated as sinking extruded pellets#4932S0 at sizes of 2-4 mm, such as 2-3 mm. Certain particular feedembodiments comprised 45.0% protein, 12.0% fat, 3.0% carbohydrates, 9%ash, and 9.8% moisture. In other particular embodiments, the feedstuffwas manufactured by Zemach Feed Mill. The feed may be formulated asfloating extruded pellets #4662 at sizes of 2-4 mm, such as 2-3 mm.Certain particular embodiments comprised 35.0% protein, 3.5% fat, 14.0%carbohydrates, 8.0% ash, and 10.0% moisture. In other particularembodiments the feed used was manufactured by Zemach Feed Mill, and wasbased on floating extruded pellets #4212 at a size of 4 mm. Certainparticular embodiments comprised 30.0% protein, 5.0% fat, 4.5%carbohydrates, 8.0% ash, and 10.0% moisture. In some embodiments, thecomposition or one or more of the components of the combination iscoated on the feedstuffs using an adhesive agent.

H. Additional Components

In some embodiments, the composition and/or combination includesadditional components. Additional components may be used for any desiredpurpose, such as a substantially biologically inert material added, forexample, as a filler, or to provide a desired beneficial effect.Alternatively or in addition, adjuvants and/or therapeutic agents alsomay be included in the composition and/or combination. For example,composition and/or combination may include, without limitation, acarbonate (including a metal carbonate such as calcium carbonate), kelp,a vitamin (such as a niacin supplement or vitamin B-12 supplement),biotin, d-calcium pantothenate, choline chloride, thiamine mononitrate,pyridoxine hydrochloride, menadione dimethylpyrimidinol bisulfite,riboflavin-5-phosphate, folic acid, soybean oil, calciumaluminosilicate, rice hulls, algae, mineral oil, or any combinationthereof. The algae may be a blue-green algae (cyanobacteria), a diatom(bacillariophyta), a stonewort algae (charophyta), a green algae(chlorophyta), a golden algae (chrysophyta), a dinoflagellate(dinophyta), a brown algae (phaeophyta) or a red algae (rhodophyta). Insome embodiments, the algae is a chlorophyta, and may be an algae fromthe genus Chlorella, including, but not limited to, Chlorella vulgaris,Chlorella angustoellipsoidea, Chlorella botryoides, Chlorella capsulata,Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorellahomosphaera, Chlorella luteo-v iridis, Chlorella marina, Chlorellaminiata, Chlorella minutissima, Chlorella mirabilis, Chlorella ovalis,Chlorella parasitica, Chlorella peruviana, Chlorella rugosa, Chlorellasaccharophila, Chlorella salina, Chlorella spaerckii, Chlorellasphaerica, Chlorella stigmatophora, Chlorella subsphaerica, Chlorellatrebouxioides, or a combination thereof. In other embodiments, the algaeis a cyanobacteria, such as Arthrospira platensis or Arthrospira maxima(spirulina). Other algae include, but are not limited to, algae of thegenus Pediastrum, such as Pediastrum dupl, Pediastrum boryanum, or acombination thereof, algae of the genus Botryococcus, such asBotryococcus braunii, algae of the genus Porphyra, such as Porphyradioica, Porphyra linearis, Porphyra lucasii, Porphyra mumfordii,Porphyra purpurea, Porphyra umbilicalis, or a combination thereof.

I. Further Compositions and/or Combinations

In some embodiments, components may be incorporated in differentmanners, including as a composition and/or as a combination. Forexample, the composition and/or combination may comprise a component 1selected from: 1A) glucan; 1B) silica; 1C) mineral clay; 1D) mannans;1E) polyphenol; 1F) glucan and silica; 1G) glucan and mineral clay; 1H)glucan and mannans; 1I) glucan and polyphenol; 1J) silica and mineralclay; 1K) silica and mannans; 1L) silica and polyphenol; 1M) mineralclay and mannans; 1N) mineral clay and polyphenol; 1O) mannans andpolyphenol; 1P) glucan, silica and mineral clay; 1Q) glucan, silica andmannans; 1R) glucan, silica and polyphenol; 1S) glucan, mineral clay andmannans; 1T) glucan, mineral clay and polyphenol; 1U) glucan, mannansand polyphenol; 1V) silica, mineral clay and mannans; 1W) silica,mineral clay and polyphenol; 1X) mineral clay, mannans and polyphenol;1Y) glucan, silica, mineral clay and mannans; 1Z) glucan, silica,mineral clay and polyphenol; 1AA) silica, mineral clay, mannans andpolyphenol; 1AB) glucan, silica, mineral clay, mannans and polyphenol;1AC) quillaja; 1AD) yucca; 1AE) a probiotic; 1AF) quillaja and yucca;1AG) quillaja and a probiotic; 1AH) yucca and a probiotic; 1AI)quillaja, yucca and a probiotic; 1AJ) Yucca schidigera; 1AK) Quillajasaponaria; 1AL) Bacillus coagulans; 1AM) Yucca schidigera and Bacilluscoagulans; 1AN) Quillaja saponaria and Bacillus coagulans; 1AO) Yuccaschidigera, and Quillaja saponaria; 1AP) Yucca schidigera, Quillajasaponaria and Bacillus coagulans; 1AQ) an antimicrobial; 1AR) anantibiotic; 1AS) Virginamycin; 1AT) an anticoccidial agent, for exampleSalinomycin; 1AU) an antifungal; 1AV) an antiviral; 1AW) anantiparasitic; 1AX) a vaccine; or 1AY) an adhesive agent.

The composition and/or combination may also comprise a component 2. Withrespect to the component 1 embodiments, the component 2 may be, in acombination with 1A to 1AY: 2A) quillaja; 2B) yucca; 2C) a probiotic;2D) quillaja and yucca; 2E) quillaja and a probiotic; 2F) yucca and aprobiotic; 2G) quillaja, yucca and a probiotic; 2H) Yucca schidigera;21) Quillaja saponaria; 2J) Bacillus coagulans; 2K) Yucca schidigera andBacillus coagulans; 2L) Quillaja saponaria and Bacillus coagulans; 2M)Yucca schidigera, and Quillaja saponaria; 2N) Yucca schidigera, Quillajasaponaria and Bacillus coagulans; 2O) an antimicrobial; 2P) anantibiotic; 2Q) Virginamycin; 2R) an anticoccidial agent, for exampleSalinomycin; 2S) an antifungal; 2T) an antiviral; 2U) an antiparasitic;2V) a vaccine; or 2W) an adhesive agent.

A person of ordinary skill in the art will understand that any of 2A to2W may be combined with any of 1A to 1AY, to form any and allcompositions and/or combinations between such substituents.

The composition and/or combination may comprise a component 3. Withrespect to the component 1 embodiments 1A to 1AY and the component 2embodiments 2A to 2W, component 3 may be, in combination with 1A to 1AYand 2A to 2W: 3A) an antimicrobial; 3B) an antibiotic; 3C) Virginamycin;3D) an anticoccidial agent, for example Salinomycin; 3E) an antifungal;3F) an antiviral; 3G) an antiparasitic; 3H) a vaccine; or 3I) anadhesive agent.

A person of ordinary skill in the art will understand that any of 3A to3I may be combined with any of 1A to 1AY and any of 2A to 2W, to formany and all compositions and/or combinations between such substituents.

The composition and/or combination may further comprise a component 4.With respect to the component 1 embodiments 1A to 1AY the component 2embodiments 2A to 2W, and the component 3 embodiments 3A to 3I,component 4 may be, in combination with 1A to 1AY, 2A to 2W, and 3A to3I: 4A) an antimicrobial; 4B) an antibiotic; 4C) Virginamycin; 4D) ananticoccidial agent, for example Salinomycin; 4E) an antifungal; 4F) anantiviral; 4G) an antiparasitic; 4H) an adhesive agent;

A person of ordinary skill in the art will understand that any of 4A to4H may be combined with any of 1A to 1AY, any of 2A to 2W, and any of 3Ato 3I, to form any and all compositions and/or combinations between suchsubstituents.

The composition and/or combination may further comprise a component 5.With respect to the component 1 embodiments 1A to 1AY, the component 2embodiments 2A to 2W, the component 3 embodiments 3A to 3I, and thecomponent 4 embodiments 4A to 4H, component 5 may be, in combinationwith 1A to 1AY, 2A to 2W, 3A to 3I, and 4A to 4H: 5A) an antimicrobial;5B) an antibiotic; 5C) Virginamycin; 5D) an anticoccidial agent, forexample Salinomycin; 5E) an antifungal; 5F) an antiviral; 5G) anantiparasitic.

A person of ordinary skill in the art will understand that any of 5A to5G may be combined with any of 1A to 1AY, any of 2A to 2W, any of 3A to3I, and any of 4A to 4H to form any and all compositions and/orcombinations between such substituents.

J. Exceptions

In any of the above embodiments, one or more of the following exceptionsmay apply.

If the composition and/or combination is, consists of, or consistsessentially of yucca and/or quillaja; and an antibiotic, anantimicrobial, an anticoccidial agent, or a combination thereof, or ifthe composition and/or combination is, consists of, or consistsessentially of yucca and/or quillaja; an antibiotic, an antimicrobial,an anticoccidial agent, or a combination thereof; and a vaccine, thenthe fish is not salmon, trout, cod, halibut, snapper, herring, orcatfish; the crustacean is not lobster, shrimp, prawns, crabs, hill,crayfish, barnacles, or copepods; and the mollusk is not abalone,conchs, rock snails, whelk, clams, oysters, mussels, or cockles.

If the composition and/or combination is, consists of, or consistsessentially of silica, mineral clay, glucan and mannans, and one or moreof yucca, quillaja, a direct-fed microbial, a vitamin D species, or aplant extract, then the fish is not salmon, trout or tilapia.

In some embodiments, the antibiotic is not, or does not comprise,hydrogen peroxide.

In some embodiments, the composition and/or combination does notcomprise a peroxide compound.

In some embodiments, the composition and/or combination does notcomprise hydrogen peroxide.

In some embodiments, the composition and/or combination does notcomprise carbamide peroxide.

In some embodiments, the composition and/or combination does notcomprise urea.

In some embodiments, the composition and/or combination does notcomprise hydrogen peroxide and urea.

III. Methods for Making

The composition and/or components of the combination may be formulatedin any suitable form, including a powder, a granule, a pellet, asolution, or a suspension. In one embodiment, the composition and/orcomponents of the combination are dry, free-flowing powder(s) suitablefor direct inclusion into a commercially-available feed, food product oras a supplement to a total mixed ration or diet. The powder may be mixedwith either solid or liquid feed or with water. In another embodiment,the composition and/or components of the combination can be formed intopellets.

In some embodiments the composition and/or combination may be a powdertop coated onto a feedstuff using an adhesive agent. In some embodimentsthe feed is mixed with adhesive agent in a mixer. The composition isadded to feedstuff and mixed until all components are suitably blended.FIG. 57 provides a schematic cross sectional view of one embodiment of aparticle comprising a feedstuff, adhesive agent and the compositionand/or combination. With reference to FIG. 57, feedstuff 2 is at leastpartially coated with adhesive agent 4, and may be substantiallycompletely coated with the adhesive agent 4. The disclosed compositionand/or combination 6 forms at least a partial layer around the particleon the adhesive agent 4, and may substantially completely cover thefeedstuff particle 2.

In some embodiments, the combination and/or composition was admixed witha feedstuff. In certain embodiments the combination and/or compositionis formulated to be suitable to form a homogeneous mixture with thefeedstuff, such as by crushing, crumbling, grinding or otherwise sizingthe combination. Alternatively, the combination and/or composition maybe formulated as a solution, suspension or slurry. In embodiments wherethe combination comprises two or more components, the components may beformulated separately or substantially together. The components may alsobe admixed with the feedstuff sequentially, in any order, orsubstantially simultaneously.

IV. Methods for Using

Embodiments of the disclosed composition and/or combination can beadministered to aquatic animals to obtain one or more beneficialresults. For example, embodiments of the composition and/or combinationmay be used to prevent and/or treat certain aquatic diseases.Additionally, the composition and/or combination may improve the feedconversion rate of an aquatic animal. A feed conversion rate, also knownas a feed conversion ratio, is a measure of an animal's efficiency inconverting feed mass into increased body mass. Animals with low feedconversion rates are considered efficient, as they require less feed toreach a desired weight. For example, tilapia typically have a feedconversion ratio of from 1.6 to 1.8, and farm raised salmon typicallyhave a ratio of around 1.2. In some embodiments the feed conversion ratemay be enhanced by administering the composition and/or combination byfrom 0.5% to 20% or more, such as from 1% to 20%, preferably from 2% to10%, and in certain embodiments, from 3% to 5%.

For some embodiments, such as with aquatic animals, the compositionand/or combination can be administered based on body weight, such asgrams of the composition and/or combination per pound or kilogram bodyweight of fish per day, or in milligrams of the composition and/orcombination per pound or kilograms of body weight. In a particularexample, when administered to fish the composition and/or combinationmay be provided in a range of from greater than zero to 500 mg per kg ofbody weight per day, such as from 10 mg to 350 mg per kg of body weightper day or from 50 mg to 250 mg per kg of body weight per day. Incertain embodiments, the composition and/or combination is administeredto the aquatic species in an amount of from 50 mg to 200 mg per kg bodyweight per day, from 50 mg to 175 mg per kg body weight per day, from 50mg to 150 mg per kg body weight per day, or from 75 mg to 125 mg per kgbody weight per day.

Alternatively, the composition and/or combination is administered basedon the amount of feed provided to the aquatic animals. In someembodiments, the amount of the composition and/or combination providedto the aquatic animals is from greater than zero to 10,000 mg per kg offeed or more, such as from 500 mg to 7,500 mg per kg of feed, or from1,000 mg to 5,000 mg per kg of feed.

A person of ordinary skill in the art will appreciate that the amount ofthe composition and/or combination administered can vary depending upona number of factors, including the animal species, size of the animal,the age or growth stage of the animal, and type of the feedstuff towhich the combination is added. In some embodiments, 100 mg per kg ofbody weight per day is administered, and in other embodiments, 200 mgper kg of body weight per day is administered. In certain embodiments,1,000 mg, 2,000 mg or 4,000 mg per kg of feed is administered to theanimals.

The composition and/or combination may be administered to the aquaticspecies at any time period during its lifetime. The composition and/orcombination may be administered throughout the aquatic species'lifetime, such as from birth or hatching to death, or it may beadministered during certain times during the lifetime, including, butnot limited to, hatchery, nursery, grow-out stages and/or up to harvest.The composition and/or combination may be continuously administered tothe aquatic species, such as continuously administered throughout thelifetime of the aquatic species, or it may be intermittentlyadministered, such as only during certain growth, developmental and/orlife stages.

FIG. 1 provides exemplary ranges for fish for hatchery, nursery andgrow-out stages, based on an administration amount of 100 mg of thecomposition and/or combination per kg of body weight per day. FIG. 1illustrates that hatchery stage fish being fed at a feeding rate of 10%of body weight per day and being administered 100 mg of the compositionand/or combination per kg of body weight per day, the dose of thecomposition and/or combination is 1,000 mg per kg of feed. Thisincreases to 2,000 mg per kg of feed for fish at the nursery stage, andup to 4,000 mg per kg of feed for fish at the grow-out stage. FIG. 1also provides exemplary feed sizes, of from greater than zero to 1 mmand from 1 mm to 2 mm for the hatchery stage, from 2 mm to 3 mm for thenursery stage, and 3 mm or greater for the grow-out stage. The feed sizemay vary depending on the species of aquatic animal as well as on thegrowth stage of the animal. Suitable feed sizes for particular aquaticanimals at different growth stages are known to persons of ordinaryskill in the art.

In particular disclosed embodiments, the composition and/or combinationmay be administered to aquatic animals using a carrier and/or adhesiveagent. The carrier and/or adhesive agent may be any carrier and/oradhesive agent known to a person of ordinary skill in the art as beingsuitable for combining with a feed composition and/or combination. Inother particular disclosed embodiments, the composition and/orcombination may be administered to the aquatic animals using adispersant or adhesive agent allowing the composition and or combinationto be coupled to the animal feedstuffs in an aquatic environment. Insome embodiments, no carrier or adhesive agent is necessary, and/or thecomposition and/or combination may be administered as a primaryfeedstuff.

The animal may be an aquatic animal, including but not limited to afish, crustacean, and mollusk. In some embodiments, the aquatic animalis a fish or a mollusk. In other embodiments, the aquatic animal is nota crustacean. Aquatic animals may be raised for consumption, ornamentaluses, or for other reasons.

The fish may be any fish, with exemplary particular species includingtilapia, such as Nile tilapia, blue tilapia, Mozambique tilapia,tilapiine cichlids, or hybrids thereof; sea bream, such as sheepshead,scup, yellowfin bream, gilt-head bream, Saucereye porgies, red seabream, or hybrids thereof; carp, such as common carp, Asian carp, Indiancarp, black carp, grass carp, silver carp, bighead carp, or hybridsthereof; salmon, such as pink salmon, chum salmon, sockeye salmon, cohosalmon, Atlantic salmon, chinook salmon, masu salmon or hybrids thereof;trout, such as rainbow trout, Adriatic trout, Bonneville cutthroattrout, brook trout, steelhead trout or hybrids thereof; cod, such asAtlantic northeast cod, Atlantic northwest cod, Pacific cod, or hybridsthereof; halibut, such as Pacific halibut, Atlantic halibut, or hybridsthereof; snapper, such as red snapper, bluefish or hybrids thereof;herring, such as Atlantic herring or Pacific herring; catfish, such aschannel catfish, walking catfish, shark catfish, Corydoras, basa, banjocatfish, talking catfish, long-whiskered catfish, armoured suckermouthcatfish, blue catfish, or hybrids thereof; flounder, such as gulfflounder, southern flounder, summer flounder, winter flounder, Europeanflounder, olive flounder, or hybrids thereof; hake, such as Europeanhake, Argentine hake, Southern hake, offshore hake, benguela hake,shallow-water hake, deep-water hake, gayi hake, silver hake, NorthPacific hake, Panama hake, Senegalese hake, or hybrids thereof; smelt;anchovy, such as European anchovy, Argentine anchoita, Californiananchovy, Japanese anchovy, Peruvian anchovy, Southern African anchovy,or hybrids thereof; lingcod; moi; perch, such as yellow perch, balkhashperch, European perch, or hybrids thereof; orange roughy; bass, such asEuropean sea bass, striped bass, black sea bass, Chilean sea bass,spotted bass, largemouth sea bass, Asian sea bass, barramundi, orhybrids thereof; tuna, such as yellowfin tuna, Atlantic bluefin tuna,pacific bluefin tuna, albacore tuna, or hybrids thereof; mahi; mackerel,such as Atlantic mackerel, Short mackerel, Blue mackerel, chub mackerel,king mackerel, Atlantic Spanish mackerel, Korean mackerel, or hybridsthereof; eel, such as American eel, European eel, Japanese eel,short-fin eel, conga eel, or hybrids thereof; barracuda, such as greatbarracuda, Pacific barracuda, Yellowstripe barracuda, Australianbarracuda, European barracuda, or hybrids thereof; marlin, such asAtlantic blue marlin, black marlin, or hybrids thereof; mullet, such asred mullet, grey mulletor hybrids thereof; Atlantic ocean perch; Nileperch; Arctic char; haddock; hoki; Alaskan pollock; turbot; freshwaterdrum; walleye; skate; sturgeon, such as beluga, Kaluga, starlet, orhybrids thereof; Dover sole or Microstomus pacificus; common sole;wolfish; sablefish; American shad; John Dory; grouper; monkfish;pompano; lake whitefish; tilefish; wahoo; cusk; bowfin; kingklip; opah;mako shark; swordfish; cobia; croaker. In certain embodiments, the term‘fish’ does not include salmon or trout. In other embodiments, the fishis selected from tilapia, sea bream, carp, cod, halibut, snapper,herring, catfish, flounder, hake, smelt, anchovy, lingcod, moi, perch,orange roughy, bass, tuna, mahi, mackerel, eel, barracuda, marlin,Atlantic ocean perch, Nile perch, Arctic char, haddock, hoki, AlaskanPollock, turbot, freshwater drum, walleye, skate, sturgeon, Dover sole,common sole, wolfish, sablefish, American shad, John Dory, grouper,monkfish, pompano, lake whitefish, tilefish, wahoo, cusk, bowfin,kingklip, opah, mako shark, swordfish, cobia, croaker, or hybridsthereof.

The composition and/or combination may be provided to any crustacean,including, but not limited to, shrimp, such as Chinese white shrimp,pink shrimp, black tiger shrimp, freshwater shrimp, gulf shrimp, Pacificwhite shrimp, whiteleg shrimp, giant tiger shrimp, rock shrimp, Akiamapaste shrimp, Southern rough shrimp, fleshy prawn, banana prawn,Northern prawn, or hybrids thereof; crab, such as blue crab, peekytoecrab, spanner crab, Jonah crab, snow crab, king crab, stone crab,Dungeness crab, soft-shell crab, Cromer crab, or hybrids thereof;lobster, such as American lobster, spiny lobster, squat lobster, orhybrids thereof; crayfish; krill; copepods; barnacles, such as goosebarnacle, picoroco barnacle, or hybrids thereof. In other embodiments,the crustacean is not a shrimp, and/or is selected from crab, lobster,crayfish, hill, copepods, barnacles, or hybrids thereof.

The mollusk may be selected from squid, such as common squid, Patagoniansquid, longfin inshore squid, neon flying squid, Argentine shortfinsquid, Humboldt squid, Japanese flying squid, Wellington squid, orhybrids thereof; octopus, such as the common octopus; clams, such ashard clam, soft-shell clam, ocean quahog, surf clam, Asari, Hamaguri,Vongola, Cozza, Tellina, or hybrids thereof; oysters, such as Pacificoyster, rock oyster, European flat oyster, Portuguese oyster, or hybridsthereof; mussel, such as blue mussel, freshwater mussel, green-lippedmussel, Asian green mussel, Mediterranean mussel, Baltic mussel, orhybrids thereof; abalone; conchs; rock snails; whelks; cockles; orcombinations thereof.

Embodiments of the compositions and/or combinations disclosed herein canbe used for feeding animals and can provide additional nutritionalbenefit to the animals to increase the feed conversion rates, to helpsupport and/or maintain the animals' overall health and well-being, suchas by helping increase longevity of the animal, helping boost immunityto disease, and other benefits.

A. Use in Prevention/Treatment of Disease

In some embodiments, the compositions and/or combinations can be used tohelp promote health in an animal at risk of developing a disease. Insome embodiments, the animal can be affirmatively selected based on oneor more factors that include the animal's age, decreased immunity,exposure to stressors or stress events (e.g., heat stress, crowding,ammonia toxicity, work load, chemotherapy, anti-inflammatory therapy),gastrointestinal disturbances (e.g., diarrheal diseases), orcombinations thereof. In exemplary embodiments, the animal can be anaquatic animal susceptible to an environmental malady, such as an acutetoxicity of ammonia due to the surrounding environment, or heat stresscaused by, for example, an elevated or reduced water temperature.Ammonia toxicity may occur when an aquatic animal is exposed to anenvironment with ammonia concentrations of greater than about 2.0 mg/L.In some embodiments the composition and/or combination is administeredprior to the animal experiencing ammonia toxicity, and/or while theanimal is experiencing ammonia toxicity. In some embodiments thecomposition and/or combination is administered prior to the animalexperiencing heat stress, and/or while the animal is experiencing heatstress. In other embodiments, the method can be used to ameliorate signsor symptoms of disease in an animal that is suffering or afflicted witha disease. Exemplary embodiments can comprise administering thecombination to an animal to help ameliorate signs or symptoms of bothinfectious and non-infectious diseases or conditions.

Examples may include the following:

-   -   Infectious disease such as Bacteria, Viruses, Fungal agents or        toxic Algae;    -   Environmental disease such as ammonia toxicity, nitrite        toxicity, nitrate toxicity, hypoxia, increased levels of        suspended solids, changes in salinity levels, hypothermia,        hyperthermia or changes in pH levels;    -   Nutritional disease such as Vitamin deficiencies, mycotoxins or        rancid feed; and    -   Genetic disease such as anatomical disorders, lordiosis or        aplasia of fins.

Stress is a condition in which an aquatic species is unable to maintaina normal physiologic state because of various factors adverselyaffecting its well-being. Some of the more common stress factors inducedin aquaculture include:

Chemical stressors, for example, poor water quality such as lowdissolved oxygen or improper pH; pollution such as intentionalpollution, chemical treatments, accidental pollution, insect spray, orspills; diet composition, such as the type of protein or amino acids;and nitrogenous and other metabolic wastes, such as accumulation ofammonia, nitrate or nitrite;

Biological stressors, for example, population density such asovercrowding; other species of fish resulting in issues of aggression,territoriality and/or lateral swimming space requirements;micro-organisms, such as pathogenic and non-pathogenic organisms; andmicro-organisms, such as internal and external parasites;

Physical stressors, for example, temperature, such as hypothermia andhyperthermia—this is one of the most important influences on the immunesystem of fish; light; sounds; and dissolved gases; and

Procedural stressors, for example, handling; shipping; and diseasetreatments.

In some embodiments, a method of administering a composition and/orcombination comprising glucan, silica, mineral clay and mannans to ananimal does not include administering the composition and/or combinationto an animal that is experiencing a stress event or stressor, and/or isat risk of experiencing a stress event or stressor. In otherembodiments, the method of administering the composition and/orcombination to an animal does not include administering the compositionand/or combination to an animal that is experiencing, or is at risk ofdeveloping heat-induced stress or heat stress.

In some embodiments, a method of administering a composition and/orcombination comprising glucan, silica, mineral clay and mannans to afish does not include administering the composition and/or combinationto a fish that is experiencing a stress event or stressor, and/or is atrisk of experiencing a stress event or stressor.

In some embodiments, a stress event does not include heat-induced stressor heat stress, such as hyperthermia or hypothermia. In otherembodiments, the environmental disease or condition is selected fromammonia toxicity, nitrite toxicity, nitrate toxicity, hypoxia, increasedlevels of suspended solids, changes in salinity levels, or changes in pHlevels.

In some embodiments, an animal administered the composition and/orcombination does not have a decreased serum cortisol level relative toan animal not fed the composition and/or combination. In otherembodiments, a fish administered the composition and/or combination doesnot have a decreased serum cortisol level relative to a fish not fed thecomposition and/or combination.

Animals disclosed herein can exhibit a response, or a combination ofresponses, to the compositions and/or combinations (or to componentsthereof) disclosed herein. In some embodiments, these responses can bedetected and measured to determine whether an animal's health issupported by administration of the composition and/or combination to theanimal. In particular disclosed embodiments, one or more factors (orendpoints) can be used to determine an animal's response to thecomposition and/or combination. In some embodiments, a factor that canbe examined is the ability of the composition and/or combination toincrease expression of markers of innate immunity.

In some embodiments, the composition and/or combination (or a componentthereof) may modify nutrient transport, and/or bind pathogenic bacteria.In another embodiment, the composition and/or combination (or acomponent thereof) may act as an emulsifier by dispersing molecules,thereby facilitating nutrient transport and/or increase the exposure ofantigens to antigen-sensing cells in an animal's gut, including, but notlimited to, M cells. In yet other embodiments, anti-oxidant,anti-inflammatory, anti-microbial, and/or anti-hypertensive propertiesof the composition and/or combination (or a component thereof) can befactors that are examined. In some embodiments, the ability of thecomposition and/or combination to beneficially affect immune modulation,metabolic regulation, nutrient utilization and/or transport, endocrineand neuroendocrine regulation, and longevity (or lifespan) can bedetermined.

An animal that is experiencing a stress event, or stressor, may have anelevated serum cortisol level, relative to an animal not experiencing astress event. In some embodiments, administering the composition and/orcombination to the animal that is experiencing a stress event maypromote a reduction in the serum cortisol level relative to an animalthat is experiencing a stress event but is not being administered thecomposition and/or combination.

The age of the animal that is to be administered the composition and/orcombination can vary with the species. Therefore, the point at which thecombination is administered can be guided by the type of animal to whichthe combination is being administered.

In some embodiments, the composition and/or combination can beadministered daily to the animal at time intervals believed ordetermined to be effective for achieving a beneficial result. Thecomposition and/or combination can be administered in a single dosedaily or in divided doses throughout the day. In some instances, one ormore individual combinational components or compositions thereofdisclosed herein may be administered to the animal at a first time, andremaining combinational components or compositions thereof may beadministered individually or in combination at one or more subsequenttimes during the same day.

In some embodiments the method comprises administering a combinationcomprising a first component and a second component and/or an additionalcomponent. The first component can comprise glucan, silica, mineralclay, mannans, polyphenol or a combination thereof. In certainembodiments, the first component comprises, consists essentially of, orconsists of, glucan, silica, mineral clay and mannans. In otherembodiments, the first component comprises, consists essentially of, orconsists of, glucan, silica, mineral clay, mannans, andendoglucanohydrolase. In further embodiments, the first componentcomprises, consists essentially of, or consists of, polyphenol. Thesecond component can comprise yucca, quillaja, or a combination thereof.In some embodiments, the second component comprises Yucca schidigera,Quillaja saponaria, or a combination thereof. The second component mayalso comprise a probiotic, such as a Bacillus species. In someembodiments, the second component comprises Bacillus coagulans, and incertain embodiments, the second component comprises, consistsessentially of, or consists of, Yucca schidigera, Quillaja saponaria andBacillus coagulans. The additional component can comprise anantimicrobial, an antibiotic, an antifungal, an antiparasitic such as ananticoccidial agent, a vaccine, or a combination thereof. In someembodiments, the additional component comprises Virginiamycin,Salinomycin, or a combination thereof. The amount of the antibiotic oranticoccidial agent in the second component can range from 10 ppm to 70ppm, with some embodiments comprising from 10 ppm to 30 ppmVirginiamycin and/or at least 25 ppm to 90 ppm Salinomycin, such as 20ppm to 80 ppm, 20 ppm to 70 ppm, 20 ppm to 60 ppm, or 20 ppm to 50 ppm.Exemplary amounts in certain working embodiments include but are notlimited to, 22 ppm Virginiamycin and 50 ppm to 70 ppm, such as 66 ppmSalinomycin. Any of the above components may also comprise an adhesiveagent.

Method embodiments disclosed herein also can comprise administering thecomposition and/or combination comprising the first component and thesecond composition in combination with a feedstuff. For example, thecombination of the first component and the second and/or thirdcomponents can be administered in combination with an amount offeedstuff suitable for obtaining an animal having a weight suitable forthat particular species. In some embodiments, the amount of feedstuffthat is provided to the animal can be varied according to their foodintake needs as growth occurs.

In some embodiments, the composition and/or combination can comprise afirst component comprising silica, glucan, mannans and mineral clay, asecond component comprising Yucca schidigera, and Quillaja Saponaria, athird component comprising an antimicrobial agent and/or an antibiotic,and a fourth component comprising a vaccine. A feedstuff may also beadministered in such embodiments. The combination of the first, second,third and/or fourth components that are administered can be admixed witha feedstuff prior to administration to the animal, or the feedstuff maybe administered before or after the combination of the first, second,third and/or fourth components. These embodiments are not intended tolimit the order of administration, as any suitable order ofadministration can be selected.

The combination and/or composition embodiments disclosed herein can beadministered using any suitable technique. In some embodiments, thecombination and/or the composition is orally administered by activelyintroducing the composition and/or combination into the animal's mouth,or orally administered by allowing the aquatic animal to ingest thecomposition and/or combination on its own. The composition and/orcombination may be administered to the animal during any stage of itslifecycle during which it consumes food.

Method embodiments disclosed herein improve an animal's feed conversionrate, such as by reducing the animal's feed conversion rate value,relative to animals that are fed a standard diet (e.g., a feedstuff). Inan independent embodiment, the method described herein can be used toimprove an animal's feed conversion rate relative to animals that aresolely fed a feedstuff in combination with amounts of a compositionand/or combination comprising Yucca schidigera and Quillaja saponariaranging from 100 ppm to 150 ppm. In some embodiments, the animal is ananimal raised for consumption. A feed conversion rate (feed conversionratio) is a measure of an animal's efficiency in converting feed massinto increased body mass.

B. Improving Feed Conversion Rates

A feed conversion rate, also known as a feed conversion ratio, is ameasure of an animal's efficiency in converting feed mass into increasedbody mass. Animals with low feed conversion rates are consideredefficient, as they require less feed to reach a desired weight. Forexample, tilapia typically have a feed conversion ratio of from 1.6 to1.8, and farm raised salmon typically have a ratio of around 1.2.

In some embodiments the feed conversion rate may be enhanced by from0.5% to greater than 20%, preferably by about 2% to about 10%, and incertain embodiments, from about 3% to 5%.

In some embodiments, a composition and/or combination of quillaja and/oryucca with a probiotic, such as Bacillus coagulans, has a beneficialeffect on animal health, typically a beneficial effect on the digestivesystem, including the stomach and intestines. Certain embodiments have abeneficial effect on villi health. Improved digestive health may causeimprovement of a feed conversion ratio.

V. Effect on Immune System Biomarkers

Composition and/or combination embodiments disclosed herein, whenadministered to an animal, may produce a concomitant change in a levelof, for example, an immune system biomarker or an inflammation biomarkerin the animal by at least 5%, at least 10%, at least 20%, at least 30%,at least 50%, at least 75%, at least 100%, at least 200%, or at least500%, such as from 5-600%, from 10-500%, from 10-200%, or from 10-100%,compared to an average level of the biomarker in an animal that has notreceived the combination. The change may be an increase or a decrease,depending on the particular biomarker.

In some embodiments, administration of the composition and/orcombination may produce a concomitant change in a level of innatedefense mechanisms of fish prior to exposure to a pathogen, or improvesurvival following exposure to a specific pathogen. Markers of improvedinnate immune response may include:

1. Total Leucocyte Count

Abnormal changes in total and differential blood cell counts in fish,such as anaemia, leukopaenia, leukocytosis and thrombocytopaenia, mayresult from diseases, but may also indicate stress, toxic exposure,hypoxia and changes in reproductive status.

Due to the nucleated nature of red blood cells (erythrocytes) in fish,white blood cells (leukocytes), which serve as an indicator of health,cannot be distinguished using automated cell counting procedures withoutlysis of erythrocytes and are usually manually counted using ahaemocytometer. Differential leukocyte and haemocyte enumerations, whichalso serve as health indicators, are generally performed either onstained smears or with a haemocytometer in fish and crustacea,respectively. The disadvantage of manual enumeration is the statisticallimitation associated with counting between 100 to 200 cells, thetypical range in differential leukocte procedures.

Flow cytometry is an instrumental technique in which a stream ofsuspended particles is interrogated by one or more lasers. Particles areanalysed and differentiated on the basis of their light-scatteringproperties, auto- or labelled fluorescence, or a combination of both.

The major advantages of flow cytometry technology are the ability todifferentiate and enumerate several thousands of particles per second,and to physically sort multiple populations simultaneously intocollection vessels. In haematological applications, the capability toobtain accurate and precise total and 5 differential blood counts on somany more cells than practically achievable with manual methods, in afraction of the time, is thus dependent only on the ability toaccurately discriminate between cell types.

2. Respiratory Burst (Release of Superoxide Anion)

Several reactive oxygen species (ROS) are produced by fish phagocytesduring the respiratory burst. Once bacteria or fungi are engulfed byleucocytes, the host's NADPH-oxidase is activated, which in turnincreases oxygen consumption and subsequently produces ROS such assuperoxide anion (O₂ ⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH)and singlet oxygen (¹O₂). The release of superoxide anion is known asthe respiratory burst, and the ROS released and/or formed may be arebactericidal.

3. Phagocytic Index and Activity

Phagocytosis is an essential component of the non-specific immuneresponse against infectious agents in teleosts. This process involvesthe recognition and attachment of foreign particles, includingpathogens, engulfment and digestion by the phagocyte. In vitro assayshave been used for studying fish macrophage phagocytic activity, therebyproviding an avenue for evaluating immunocompetence in fish. In vitroassays have also provided insight for non-specifically enhancing diseaseresistance in finfish aquaculture, and have served as immunologicalbiomarker tests to assess aquatic environmental health.

4. Lysozyme Activity

Lysozyme found in cutaneous mucus, peripheral blood and certain tissuesrich in leucocytes, is an enzyme which catalyzes the hydrolysis ofN-acetyl muramic acid and N-acetyl glucosamine of peptidoglycan inbacterial cell walls. This protein plays a crucial role in the defensesystem.

In other embodiments, administration of the composition and/orcombination may produce a concomitant change in a level of innatedefense mechanisms of crustaceans prior to exposure to a pathogen, orimprove survival following exposure to a specific pathogen. Markers ofimproved innate immune response in crustaceans may include:

1. Total Hemocyte Count

Haemocytes play a central role in crustacean immune defense. Firstly,they remove foreign particles in the hemocoel by phagocytosis,encapsulation and nodular aggregation. Secondly, haemocytes take part inwound healing by cellular clumping and initiation of coagulationprocesses through the release of factors required for plasma gelation.

The hemogram consists of the total haemocyte count (THC) and thedifferential haemocyte count (DHC). For the DHC, most researchers agreewith the identification of three cell types in penaeid shrimp: largegranule haemocytes (LGH), small granule haemocytes (SGH) and agranularhaemocytes or hyaline cells (HC).

THC can be easily determined using a hemocytometer, whereasdetermination of DHC requires a more complex haemocyte identification.DHC can be determined by using morphological criteria such as size andshape of cells and the difference of haemocyte refractivity using aphase contrast microscope. Although this technique is rapid, it shouldbe mentioned that when using this technique it is easy to obtain largevariations in results possibly due to interpretation errors.

Different haemocyte types can be determined using cytochemical studiesof enzyme activity detection or specific stains. The results obtainedfrom cytochemical stains for penaeid shrimp indicate that these specificstainings can differentiate between the types of haemocytes and provideadditional information on their functions. An alternative method forcell identification is the use of monoclonal antibodies (mAbs) in orderto find antigenic markers of different cell types. Using mAbs againstdifferent subpopulations of haemocytes separated by isopycniccentrifugation on a Percoll gradient, it has been found in P. japonicusthat HC share epitopes with SGH, and that an antigen was specificallyexpressed for LGH. Monoclonal antibodies could be considered as powerfultools for the development of haemocyte lineages and haemocyteproliferation studies, as well as for the isolation and study of plasmacomponents.

2. Phagocytic Activity

Phagocytosis is the most common reaction of cellular defense. Duringphagocytosis, particles or microorganisms are internalized into the cellwhich later forms a digestive vacuole called the phagosome. Theelimination of phagocyted particles involves the release of degradativeenzymes into the phagosome and the generation of reactive oxygenintermediates (ROIs). This last process is known as the respiratoryburst. The first ROI generated during this process is the superoxideanion. Subsequent reactions will produce other ROIs, such as hydrogenperoxide, hydroxyl radicals and singlet oxygen. Hydrogen peroxide can beconverted to hypochlorous acid via the myeloperoxidase system, forming apotent antibacterial system.

Despite the limited number of studies focusing on respiratory burst inpenaeid shrimp, the actual results are very interesting in view of theirvalue as biomarker of environmental disturbances. Furthermore, theimportance of respiratory burst as a microbicidal mechanism in penaeidshrimp is strongly suggested by the fact that pathogenic bacteria ofshrimp have developed ways of circumventing this mechanism. In P.Íannamei, O₂ generation is not produced when virulent Vibrio Íulnificusis used as elicitor, as opposed to strong stimulation generated by V.lginolyticus and other bacteria, such as Escherichia coli.

3. Phenoloxidase (PO) and Prophenoloxidase (ProPO) Activity

The PO is responsible for the melanization process in arthropods. The POenzyme results from the activation of the ProPO enzyme. The ProPOactivating system has been very well studied in crustaceans. Using thesedifferent approaches, the function of the ProPO system can be betterunderstood in relation to the health status of shrimp. Some studies haveshown that ProPO could be used as health and environmental markersbecause changes are correlated with infectious state and environmentalvariations, this issue which has recently been confirmed also at thegene expression level. Phenoloxidase, which has been detected in a widerange of invertebrates, is activated by several microbialpolysaccharides, including β-1,3-glucan from fungal cell walls andpeptidoglycans or lipopolysaccharides from bacterial cell walls.

4. Antibacterial Activity

Antibacterial peptides and proteins have been well studied inarthropods, mainly in insects and chelicerata, where the families ofantimicrobial molecules have been isolated and characterized. Incrustacean, some studies have shown the ability of crustacean haemolymphto inhibit bacterial growth. Several antibacterial proteins, active invitro against Gram-positive and Gram-negative bacteria, were found in C.maenas.

In the literature there are reports showing that antibacterial activityin crustaceans can be considered as an environmental marker. Therefore,many researchers have developed quantitative antibacterial assays basedon inhibition of bacterial growth on agar plate (zone inhibition assayand colony-forming units (CFU) inhibition assay), or in liquid medium onmicrotiter plates (turbidometric assay), to detect the antibacterialability in crustacean haemolymph. Using the CFU inhibition technique,antibacterial activity has been found in granular haemocytes of theshore crab C. maenas and in other crustacean species. It has beenreported that a potent antibacterial activity in the serum of C.sapidus, using the zone inhibition assay and turbidimetric test. Usingthe CFU inhibition assay, bactericidal activity against Gram negativebacteria have been described in the haemolymph of P. monodon. In P.Íannamei, strong antibacterial activity of plasma against differentmarine bacteria has been observed, using a turbidimetric assay.

5. Plasma Protein Concentration

In recent years blood metabolites have been investigated as a tool formonitoring physiological condition in wild or cultured crustaceansexposed to different environmental conditions. Hemocyanin is the majorhemolymph constituent (>60%); the remaining proteins (in order ofconcentration) include coagulogen, apohemocyanin, hormones, andlipoproteins. Blood protein levels fluctuate with changes inenvironmental and physiological conditions and play fundamental roles inthe physiology of crustaceans from O₂ transport to reproduction up tostress responses. In fact, moulting, reproduction, nutritional state,infection, hypoxia, and salinity variations are the major factorsaffecting the relative proportions and total quantities of the hemolymphproteins.

The shrimp immune system response is largely based on proteins. Theseare involved for example in recognizing foreign particles and intrapping foreign invading organisms and prevent blood loss uponwounding. Recently, it has been shown that shrimp are well adapted touse protein as a source of energy and molecules. Blood proteinconcentration has been found to be related to nutritional condition in anumber of crustaceans. The concentration of protein in the blood is apossible index of nutritional condition, which decreases in starvedprawns and lobsters. The moult cycle imposes constraints on proteinlevels, blood-proteins typically drop just before moulting as water istaken up and protein is used to synthesize the new exoskeleton. Proteinlevels then gradually build up again after ecdysis as water is replacedby tissue. Consequently, measuring the blood protein concentration of acrustacean sample group can provide valuable information to identify itscondition. The concentration of protein in the blood is directlyproportional to the refractive index of the blood. Measurements of theblood refractive index therefore offer potential as a field method forassessing the nutritional condition of prawns.

Colorimetric procedures are generally the preferred choice to measureserum protein concentration; however, they are expensive, timeconsuming, and not easily performed in the field. Because of ease, rapidmode of operation, and small amount of material required, measuringserum protein concentration using a refractometer provided anondestructive field method to assess crustacean's physiological state(stress, immunoresponse, nutrition status, molt, etc.) without any needof laboratory facilities; the refractometer is a simple, small portableinstrument that can be used in the field or on crustacean farms.

VI. EXAMPLES

The following examples are provided to illustrate certain features ofworking embodiments. A person of ordinary skill in the art willappreciate that the scope of the invention is not limited to only theseparticular features.

Example 1

A. Methods

In this example, the composition and/or combination was administered asa composition comprising between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans.Embodiments of the composition were used as a feed additive for seabream. Juveniles of Gilthead sea bream (Sparus aurata) were stocked in12 tanks in the experimental station. Each tank of 1.0 cubic meters wasstocked with 55 juvenile sea bream at an average weight of 26 grams. Thewater source was from a well at a stable temperature of 21° C., with aconstant total salinity of 18.0 parts per thousand. The duration of theexperiment was 158 days.

The experimental protocol included continuous assessment for thepresence of diseases causing organisms. Growth performance parameters ofthe fish were recorded regularly. The daily/weekly assessment of waterquality parameters included ammonia, nitrite, pH, temperature andoxygen.

Feeding rate was based on the recommended commercial feeding chart ofPhibro Aqua and adjusted according to the size of the fish and the watertemperature (FIG. 8). Feeding was performed manually twice a day. Thefeeding quantity for each tank was adjusted after evaluating the averageweight of the fish in each tank every two weeks.

The composition was top-coated on the pellets using 2 wt % of soy oil asan adhesive agent. The control group was given the same feed coated with2 wt % soy oil. The feed preparation for the trial included mixing theweighted feed in a mixer for 5 minutes with 2 wt % soy oil, and thenadditional 5 minutes mixing with the composition. The experiment wascarryout in replicates of 4 tanks per treatment. In the trial 2different doses of the composition in the feed were compared: 100milligrams per kilogram of bodyweight per day; and 200 milligrams perkilogram of bodyweight per day.

The feed used in this trial was made by Raanan Fish Meal and was basedon sinking extruded pellets #4932S0 at sizes of 2-4 millimeters;containing 45.0% protein, 12.0% fat, 3.0% carbohydrates, 9% ash and 9.8%moisture.

B. Results

General Health Parameters:

1. Survival rates in all the tanks for all the treatments were high(99.1-99.5%).

2. No external or internal parasites were detected in the trial.

3. The general health condition as indicated by the vitality and theresponse to the feeding was very good for all the treatments for theentire trial.

A significantly higher growth rate of the fish fed with dose (A), using100 milligrams of the composition per kilogram of bodyweight per day wasobtained in this trial. A better growth rate in treatment (A) wasobserved by day 17. This difference became statistically significant byday 59 (FIGS. 2-4). Without being bound to a particular theory, thebetter growth rate may be due to an improved nutrition for the fishand/or improved immunostimulant ingredients in the feed.

As shown in this trial, the response of the fish to the compositionand/or combination was significantly better compared to a control groupwithout the composition and/or combination. This conclusion emphasizedthe efficacy and the advantage of the composition and/or combination asan effective feed additive in aquatic animals such as fish.

Treatment (A) had the lowest significant feed conversion ratio (FCR)value among the 3 treatments (FIG. 5). This demonstrated the advantageof the composition and/or combination as an advanced performer,improving the feed intake ability of the fish. This ability to lower theFCR value is a major factor in aquaculture management, because itreduced the feeding cost, which is often the highest cost for fish andshrimp farmers.

The environmental conditions of this example in terms of watertemperature, dissolved oxygen levels and water quality were optimal forrearing sea bream. The growth rates of all the 3 groups were accordingto the expected growth rate of sea bream. The high percentage ofsurvival (99.1-99.5%) in all 3 groups in this study emphasized theoptimum conditions during the trial (FIGS. 6-12). The lower temperatureat the end of the trial affected the optimal growth rate of the fish butstill the advantages of the composition were evident. FIGS. 13A-13Eillustrate the experimental setup.

Example 2

A. Methods

In this example, the composition and/or combination was administered asa composition comprising between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans. Thecomposition was used as a feed additive for tilapia. Juveniles of hybridtilapia (Oreochromis niloticus×O. aureus) were stocked in 18 cages inthe experimental station. The total volume of the experimental systemwas 600 cubic meters. Each cage of 1 cubic meter in volume with a 25millimeter mesh net was stocked with 35 fish at an average weight of 95grams. The water source was from a well at a stable temperature of 24°C. The duration of the experiment was 149 days.

The experimental protocol included continuous assessment for thepresence of disease causing organisms. Growth performance parameters ofthe fish were recorded regularly. The daily/weekly assessment of waterquality parameters included ammonia, nitrite, pH, temperature anddissolved oxygen.

Feeding rate was based on the recommended commercial feeding chart ofPhibro Aqua and adjusted according to the size of the fish and the watertemperature (FIG. 21). Feeding was performed manually twice a day. Thefeeding quantity for each cage was adjusted after evaluating the averageweight of the fish in each cage every two weeks.

The composition was top-coated on the pellets using 2 wt % soy oil asthe adhesive agent. The control group was given the same feed coatedwith 2 wt % soy oil. The feed for the trial was prepared by mixing theweighted feed in a mixer for 5 minutes with 2 wt % soy oil, and thenadditional 5 minutes mixing with the composition. In this trial 2different doses of the composition in the feed were compared: 100milligrams AI per kilogram of bodyweight per day; and 200 milligram AIper kilogram of bodyweight per day.

Replicates of 6 cages were used per treatment, which were dividedequally in the rearing system. The feed for this trial was manufacturedby Zemach Feed Mill. The feed is based on floating extruded pellets#4662 at sizes of 2-4 millimeters; containing 35.0% protein, 3.5% fat,14.0% carbohydrates, 8.0% ash and 10.0% moisture.

B. Results

General Health Parameters:

1. Survival rate in all the cages for all the treatments were excellent,without mortality.

2. External parasites (Trichodina and Dactylogyrus) were detected at lowincidence. The fish were treated with formalin 37% and Bromex solution(50% Naled).

3. Low presence of dignea parasite, Centrocestus, was detected. Notreatment was required.

4. The general health condition as indicated by the vitality and theresponse to the feeding was very good for all treatments for the entiretrial.

A significantly higher growth rate of the fish fed with dose (A), using100 mg of the composition per kilogram of bodyweight per day wasobtained in this trial. A better growth rate in treatment (A) wasobserved by day 16. This difference became statistically significant byday 86 (FIGS. 14-16). Without being bound to a particular theory, thebetter growth rate may be due to an improved nutrition for the fishand/or improved immunostimulant ingredients in the feed.

Administering the composition led to a better growth rate and a betterfeed intake. 100 and 200 milligrams/kilogram bodyweight per day doseswere administered. As shown in this trial, the response of the fish tothe composition was significantly better compared to the control groupwithout the composition and/or combination. This conclusion emphasizedthe efficacy and the advantage of the composition and/or combination asan effective feed additive in aquatic animals such as fish.

Treatment (A) had the lowest significant FCR value among the 3treatments (FIGS. 17-18). This demonstrated the advantage of thecomposition and/or combination as an advanced performer, improving thefeed intake ability of the fish. This ability to lower the FCR value isa major factor in aquaculture management. The feeding cost is often thehighest cost for fish and shrimp farmers.

The environmental conditions of the experiment in terms of watertemperature, dissolved oxygen levels and water quality were optimal forrearing tilapia. The growth rates of all the 3 groups were better whencompared to the expected growth rate of tilapia, emphasizing the optimalconditions of the trial (FIGS. 19-24). The high percentage of survival(100%) in all 3 groups in this study also emphasized the optimumconditions during the trial. The lower temperature at the end of thetrial affected the optimal growth rate of the fish but still theadvantages of the composition and/or combination were evident. FIGS.25A-25C show the experimental setup.

Example 3

A. Methods

In this example, the composition and/or combination was administered asa composition comprising between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans. Thecomposition was used as a feed additive for carp. Juveniles of Commoncarp (Cyprinus carpio) were stocked in 18 cages in the experimentalstation. The total volume of the experimental system was 600 cubicmeters. Each cage of 1 cubic meter in volume with a 25 millimeter meshnet was stocked with 35 fish at an average weight of 160 grams. Thewater source was from a well at a stable temperature of 24° C. Theduration of the experiment was 83 days.

The experimental protocol included continuous assessment for thepresence of diseases causing organisms. Growth performance parameters ofthe fish were recorded regularly. The daily/weekly assessment of waterquality parameters included ammonia, nitrite, pH, temperature anddissolved oxygen.

Feeding rate was based on the recommended commercial feeding chart ofPhibro Aqua and adjusted according to the size of the fish and the watertemperature (FIG. 33). Feeding was performed manually twice a day. Thefeeding quantity for each cage was adjusted after evaluating the averageweight of the fish in each cage every two weeks.

The composition was top-coated on the pellets using 2 wt % soy oil asthe adhesive agent. The control group was given the same feed coatedwith 2 wt % soy oil. The feed for the trial was prepared by mixing theweighted feed in a mixer for 5 minutes with 2 wt % soy oil, and thenadditional 5 minutes mixing with the composition. In this trial 2different doses of the composition and/or combination in the feed werecompared: 100 mg per kg of body weight per day; and 200 mg per kg ofbody weight per day.

Replicates of 6 cages were used per treatment, which were dividedequally in the rearing system. The feed for this trial was manufacturedby Zemach Feed Mill. The feed is based on floating extruded pellets#4212 at size of 4 millimeters; containing 30.0% protein, 5.0% fat, 4.5%carbohydrates, 8.0% ash and 10.0% moisture.

B. Results

General Health Parameters:

1. Survival rate in all the cages for all the treatments were excellent,without mortality.

2. External parasites (Gyrodectylus and Dactylogyrus) were detected atlow incidence. The fish were treated with formalin 37% and Bromexsolution (50% Naled).

3. The general health condition as indicated by the vitality and theresponse to the feeding was very good for all treatments for the entiretrial.

A significant higher growth rate of the fish fed with dose (A), using100 milligrams of the composition per kilogram of bodyweight per day wasobtained in this trial. A better growth rate in treatment (A) wasobserved by day 41. This difference became statistically significant byday 83 (FIGS. 26-28). Without being bound to a particular theory, thebetter growth rate may be due to an improved nutrition for the fishand/or improved immunostimulant ingredients in the feed.

Administering the composition led to a better growth rate and a betterfeed intake. 100 and 200 milligram/kilogram bodyweight per day doseswere administered. As shown in this trial, the response of the fish tothe composition was significantly better compared to the control groupwithout the composition and/or combination. This conclusion emphasizedthe efficacy and the advantage of the composition and/or combination asan effective feed additive in animals like fish.

Treatment (A) had the lowest (insignificant) FCR value among the 3treatments (FIGS. 29 and 30). This demonstrated the advantage of thecomposition and/or combination as an advanced performer, improving thefeed intake ability of the fish. This ability to lower the FCR value isa major factor in aquaculture management. The feeding cost is often thehighest cost for fish and shrimp farmers.

The temperatures of the experiment demonstrated a cold water environment(16-21° C.). This range of temperatures is common in carp cultureworldwide. These low temperatures affected the optimal growth rate ofthe fish but still the advantages of the composition and/or combinationwere evident. The water conditions in terms of dissolved oxygen levels,ammonia, nitrite and pH were optimal for rearing carp. The highpercentage of survival (100%) in all 3 groups in this study emphasizedthe optimum conditions during the trial (FIGS. 31-36). FIGS. 37A-37Cshow the experimental setup.

Example 4

A. Background

In this example, the composition and/or combination was administered asa composition comprising between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans. Thecomposition was used as an immune modulator for hybrid tilapia. Ammoniais a toxic compound that can adversely affect fish health. The natureand degree of toxicity depends on many factors, including the chemicalform of ammonia, the pH and temperature of the water, the length ofexposure, and the life stage of the exposed fish. In natural surfacewaters, ammonia occurs in two forms: ionized ammonia, NH₄ ⁺, andun-ionized ammonia, NH₃. In fish, ammonia is a byproduct of proteinmetabolism and is primarily excreted across the gill membranes, with asmall amount excreted in the urine. Ammonia's toxicity is principallydue to the un-ionized form, NH₃. As pH increases, the toxicity ofammonia rises because the relative proportion of unionized ammoniaincreases. The toxicity of ammonia may cause convulsions, coma anddeath. Without being bound to a particular theory, elevated NH₄ ⁺ in thefish body may displace K⁺ and depolarize neurons, causing activation ofglutamate receptor, which leads to an influx of excessive Ca²⁺ andsubsequent cell death in the central nervous system. In the case oflarvae of common carp, acute toxicity of 1.76 parts per million of NH₃caused 50% mortality in the group after 24 hours. Chronic effects ofammonia were studied in three batches of turbot (Scophthalmus maximus)juveniles (14, 23 and 104 grams) exposed for 4-6 weeks to constantammonium chloride solutions. Under the environmental conditions used(16.5-17.5° C., 7.92-8.03, salinity 34.5 parts per thousand, over 80%oxygen saturation), no mortalities occurred up to 0.4 parts per millionunionized ammonia. In adapted small turbot, no major physiologicaldisturbances were observed up to 0.4-0.5 parts per million, while largeturbot were more sensitive to ammonia.

The ability to improve the resistance of aquatic species to the toxicityof the ammonia has been investigated. Tiger shrimp (Penaeus monodon),5-day post larvae, were fed diets supplemented with 0 and 71.5 park permillion astaxanthin for 8 weeks, Shrimp were then subjected to 72 hoursexposure of ammonia at 0.02, 0.2, 2 and 20 parts per million. Thesurvival rates of the astaxanthin-fed shrimp were higher than those ofthe control shrimp under all levels of ammonia except 20 ppm, showingthat the shrimp's resistance to ammonia stress had been improved bydietary astaxanthin. Other research has investigated the effects ofdietary mannans oligosaccharide (MOS) on growth performance, gutmorphology, and NH₃. stress tolerance of Pacific white shrimpLitopenaeus vannamei. After NH₃ stress for 24 hours, survival rates ofshrimp fed 2.0, 4.0, 6.0 and 8.0 grams/kilogram MOS-supplemented dietswere significantly higher (P<0.05) than that of shrimp fed a controldiet.

The purpose of this study was to evaluate the effect of the compositionand/or combination on the fish resistance to the stressful condition oftoxic ammonia levels in the water.

B. Methods

Hybrid tilapia (Oreochromis niloticus×O. aureus) were stocked in 12tanks in the experimental station. Each tank of 230 liter in volume wasstocked with 10 fish with an average weight of 350 grams per fish. Thewater source was from a well with a constant water temperature of 22° C.and constant salinity of 1,300 milligrams chloride. The duration of theexperiment was 74 days. During the first phase, 6 tanks were fed 100milligrams of the composition and/or combination per kilogram ofbodyweight per day, while the other 6 tanks were fed with commercialfeed without supplement. After 30 days of feeding in optimal conditionsof water, the water inlet was reduced, allowing the water quality todeteriorate for an additional 30 days. In the third phase of 14 days thewater inlet was closed completely and ammonium chloride (NH₄Cl) wasadded to each tank on a daily basis. This phase was characterized by acontinuous mortality of the fish showing clinical symptoms of ammoniatoxicity and bacterial infections associated with poor water quality.

The experimental protocol included continuous assessment for thepresence of diseases causing organisms. The daily assessment of waterquality parameters included ammonia, nitrite, pH, water temperature anddissolved oxygen.

Feeding rate was 1% of bodyweight, based on the recommended commercialfeeding chart of Phibro Aqua and was adjusted according to the watertemperature and the response of the fish (FIG. 21). Feeding wasperformed manually twice a day. The composition was top-coated on thepellets using 2 wt % soy oil as the adhesive agent. The control groupwas given the same commercial feed coated with 2 wt % soy oil, butwithout the composition. The feed for the trial was prepared by mixingthe weighted feed in a mixer for 5 minutes with 2 wt % soy oil, and thenadditional 5 minutes mixing with the composition. The feed for thistrial was manufactured by Zemach Feed Mill. The feed is based onfloating extruded 4 mm pellets, #4662; containing 35.0% protein, 3.5%fat, 14.0% carbohydrates, 8.0% ash and 10.0% moisture.

C. Results

General Health Parameters:

1. At the third phase (14 days) the fish didn't respond to the feed.

2. The moribund and the dead fish that were collected during the trialhad typical clinical symptoms of toxicity of ammonia.

The results of this trial showed a significant higher resistant fish feda diet with the composition and/or combination at a dose of 100 mg/kg ofbody weight per day compared to the control without the compositionand/or combination (FIGS. 38-41). In this trial, the moribund and thedead fish that were collected during the trial had typical clinicalsymptoms of ammonia toxicity, including convulsions, gill necrosis,coma, and death.

Poor water quality suppresses the immune system of the fish, enablingparasites and bacteria to enter the fish body, causing disease outbreakand consequently mortality. In the experiment, the clean water inletflow was reduced to cause deterioration of the water quality, whichfinally resulted in death in the most stressed and frail fish in thistrial (FIGS. 42-49D).

Feeding tilapia with the composition and/or combination at a dose of 100milligrams/kilogram of body weight per day for 30 days periodsignificantly improved their resistance and survival under poor waterconditions such as high levels of ammonia and nitrite. FIGS. 50A and 50Bshow details of the experimental setup.

Example 5 The Composition and/or Combination as an Immune Modulator onthe Survival and the Overall Health Status of the Pacific White Shrimp(Litopenaeus vannamei) Materials and Methods General Design of theClinical Field Study

2,400 Postlarva-20 days of Litopenaeus vannamei were stocked in 12 tanksin the experimental station. Each tank of 500 liter in volume wasstocked with 200 PL-20, at an estimated weight of 0.15 g per postlarvae.The experimental unit included a central collecting tank and a centralbiofilter. The water source was from a well. Balance marine salt wasadded to the water to achieve a total salinity of 10 ppt (parts perthousand). The duration of the experiment was 71 days. The average sizeof the shrimp at the end of the trial was around 10 grams.

Diet Supplemented with the Disclosed Composition and/or Combination

In this trial, the composition and/or combination was administered as acomposition comprising between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans. 2different doses of the composition were compared to a control. 4 tankswere fed 100 mg of the composition per kg of BW per day, 4 tanks werefed 200 mg of the composition per kg of BW per day, while the other 4tanks were fed with commercial feed without supplement.

Feeding rate based on the recommended feeding chart of Phibro Aqua forShrimp. Feed quantity was adjusted according to the water temperature,the response of the shrimp and the estimation of their average weight.Feeding was performed manually twice a day. The composition wastop-coated on the pellets using 2% of Soy oil as the adhesive agent. Thecontrol group was given the same feed without supplement coated with 2%Soy oil. The feed for the trial was prepared by mixing the weighted feedin a cement mixer (maximum load of 50 kg) for 5 minutes with 2% Soy oil,and then additional 5 minutes mixing with the supplement.

General Conditions

The experimental protocol included continuous assessment for thepresence of diseases causing organisms. The daily assessment of waterquality parameters included total salinity, ammonia, nitrite, pH, watertemperature and dissolved oxygen.

Results

The results illustrate that a significantly greater percentage of theshrimp that were fed the supplement survived, compared to the controlgroup (FIG. 51). FIG. 52 provides the water quality in the experiment.The conditions of the trial were excellent for growing shrimp. Shrimphad good body condition and good coloration. No external parasites weredetected. The final average weight of the shrimp was around 10 grams,normal for in-door culture. At this stage (nursery), the survival ratesof the control groups (60%) are normal. FIG. 53 shows the experimentalsetup, and FIG. 54 shows shrimp at the end of the trial.

In a different trial, 2500 shrimp in a pond were administered acomposition comprising between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans. After 6months, the shrimp were compared to 2500 control shrimp in a separatepond that were not administered the composition. The composition-fedshrimp has an 86% survival rate, compared to 22% for the control shrimp,and also had a greater yield (kg/pond) than the control shrimp.

Example 6 The Composition and/or Combination Increased Weight Gain inShrimp

A composition comprising silica, mineral clay, yeast cell wall extract,and β-1,3 (4)-endoglucanohydrolase was fed to 2500 shrimp in an amountof 0.2% by weight of feed over 4 months. A control group of 2500 shrimpwas fed only shrimp food, but kept under the same conditions. 100 shrimpfrom each group were randomly removed each month and weighed, thenreturned to the group. The results indicated that the group that wasadministered the composition in addition to feed grew larger over thefour months of the test (Table 1).

TABLE 1 Composition Control Group Group Average shrimp weight after 1month 0.333 g  0.282 g  Average shrimp weight after 2 month  0.8 g 0.68g Average shrimp weight after 3 month 1.75 g 2.98 g Average shrimpweight after 4 month 5.19 g 7.70 g

In a second study, shrimp fed the same composition over 6 monthsdemonstrated a four-fold increase survival rate (FIG. 55) and anincrease in the shrimp yield per pond (FIG. 56).

Example 7 The Effect of the Composition and/or Combination on the GrowthPerformance, Immune Response and Overall Health Status of Nile Tilapia(Oreochromis niloticus) Challenged by Streptococcus agalactiae Objective

The objective of this trial is to investigate the effect of thedisclosed composition and/or combination on Tilapia, by comparingbetween groups of fish fed with the composition and/or combination andcontrol groups of fish with no additive in their diet. In this trial,the composition and/or combination is administered as a compositioncomprising between 15% and 40% silica, between 50% and 81% mineral clay,between 1.0% and 5.0% β-glucans, between 0.05% and 3.0% β-1,3(4)-endoglucanohydrolase and between 1% and 8.0% mannans.

Trial Design

General design—this trial is divided to two phases. In the first phase,the treated fish are fed one embodiment of the disclosed compositionand/or combination for a period of 90 days, while the control groupsreceive no additive in the diet. The growth performance parameters thatare compared in this phase are: survival, growth rate, FCR and generalhealth status.

In the second phase, the fish are challenged with S. agalactiae,generating LD50-70 in the groups. The parameters that are detected inthis phase will indicate the immune response. This phase will becompleted after 3 continuous days with no mortality in any group.

Fish—the fish used in the trail are Nile tilapia (Oreochromisniloticus). The initial average weight of the fish is 20-100 g. The fishare naive for S. agalactiae. The fish are checked for health andpresence of parasite before the trial. Only healthy fish are used forthe trial. All the groups are acclimated for 2 weeks in the testing unitbefore the trial and are treated if necessary.

Testing unit and water condition—the testing unit is based on tanks orcages. The trial includes 5-6 replicates per treatment. Each unitincludes at least 30 fish per unit. Water conditions are in the optimalrange for Tilapia culture: maximum salinity of 2 ppt, temperature rangeof 27-30° C., oxygen level higher than 6 ppt, and a pH of 7-8.

Weighing and feeding—weighing is done every 2 weeks. All the fish in thetank/cage are sampled. The feeding rate and the size of the pellets areadjusted based on the average weight and the recommended feeding amountsof the composition and/or combination. The feed is served to the fish2-3 times a day. The amount of the composition and/or combination thatis added to the feed is based on the recommended inclusion rate. It isadded to the feed by top-coating with 2% of oil or by integrating itinto the feed in the pelleting phase.

Monitoring—water quality is measured daily for oxygen and temperatureand weekly for ammonia and nitrite. And the water is analyzed forexternal parasites every 2 weeks.

Immune response—at the end of the trial the following parameters aremeasured:

1. Blood count and non-specific immune response—erythrocyte (RBC),leukocyte (WBC), hematocrit (Hct), haemoglobin (Hb) amount, leukocytecell types (percentage of lymphocyte, monocyte, neutrophil, eosinophil)blood cell sizes, and the values of phagocytic activities anderythrocyte cell indexes (MCV and MCH) are measured.

2. Liver health—determined by comparing the size and the color of theliver of 5 fish per tank/cage. The livers pathology is also analyzed bycomparing the health status by using histology slides; and

3. Gene expression—selected genes for immune response are analyzed inthis study. The gene expression is determined at the end of the trial bycomparing the treated and the non-treated fish.

Example 8 The Effect of Composition and/or Combination on the GrowthPerformance and the Overall Health Status of Tilapia (Oreochromis spp.)Compared to its Main Ingredients (β-Glucans and Mannans, Silica andClay) Objective

The objective of this trial is to investigate the effect of thedisclosed composition and/or combination on Tilapia, by comparingbetween groups of fish fed with the composition and/or combination,β-glucans and mannans, silica, clay and a control group of fish with noadditive in their diet. In this trial, the composition and/orcombination is administered as a composition comprising between 15% and40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0%β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase andbetween 1% and 8.0% mannans.

The parameters that are collected in this trial includes: growth rate,survival, FCR (feed conversion ratio), blood count and liver health.

Materials and Methods

Testing unit and water condition—the testing unit is based on tanks orcages. The trial includes 5-6 replicates per treatment, and a total of25-30 units for the entire trial. Each unit will include at least 30fish per unit. Water conditions are in the optimal range for Tilapiaculture, i.e., maximum salinity of 2 ppt, temperature range of 27-30°C., oxygen level higher than 6 ppt, and a pH of 7-8.

Treatment groups—the trial includes 4 treatments with different feedadditives and one control group with no additive in the diet. Thetreatments and the inclusion rates will be:

1. the composition and/or combination at inclusion rate of 5 g/kg feed;

2. β-glucans and mannans at an inclusion rate of 1 g/kg feed;

3. silicon dioxide at an inclusion rate of 2 g/kg feed;

4. clay at an inclusion rate of 3.5 g/kg feed; and

5. control with no additive.

Feed preparation and feeding—the additives are added to the feed bytop-coating with 2% of soy oil or by integrating it into the feed in thepelleting phase. The feed for the trial is based on standard commercialfeed for Tilapia. The control group is fed the same feed with anadditional of 2% of soy oil by top-coating. The feed is served to thefish 2-3 times a day.

Fish—fish are Tilapia (Oreochromis spp.). The initial average weight ofthe fish is 20-100 g. The fish are checked for health and presence ofparasites before the trial. Only healthy fish are used for this trial.All the groups are acclimated for 2 weeks in the testing unit before thetrial and are treated if necessary.

Weighing—weighing is done every 2 weeks. All the fish in the tank/cageare sampled. The feeding rate and the size of the pellets are adjustedbased on the average weight and the recommended feeding amounts for thecomposition and/or combination.

Monitoring—water quality is measured daily for oxygen and temperatureand weekly for ammonia and nitrite. And the water is analyzed forexternal parasites every 2 weeks.

Non-specific immune response—at the end of the trial, the followingparameters will be measured:

1. Blood count—erythrocyte (RBC), leukocyte (WBC), hematocrit (Hct),haemoglobin (Hb) amount, leukocyte cell types (percentage of lymphocyte,monocyte, neutrophil, eosinophil) blood cell sizes.

2. Liver health—determined by comparing the size and the color of theliver of 5 fish per tank/cage. The livers pathology is also analyzed bycomparing the health status by using histology slides.

Example 9 The Effects of the Composition and/or Combination on ShrimpHealth and Survival and Challenged with a Known Pathogen ExperimentalDesign Experimental Feed

The disclosed composition and/or combination is incorporated into thefeed (e.g. Rangen shrimp feed) at an inclusion rate of from 0.2% to 0.5%or more, depending on shrimp size. In this trial, the composition and/orcombination is administered as a composition comprising between 15% and40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0%β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase andbetween 1% and 8.0% mannans.

Methodology

After an acclimation period to experimental conditions, the embodimentof the composition and/or combination is provided to five replicatetanks at 3% of biomass per day divided in two rations. During thefeeding period, the control treatment has ten aquaria, but subsequentlythey are divided into positive (infected) and negative (non-infected)control treatments. The feed for the control is a re-pelleted commercialfeed with no additive throughout the trial. The shrimp are weighedbefore and after the feeding period. After a 21 day feeding period theshrimp are challenged with a defined disease (e.g. white spot syndrome(WSS) or acute hepatopancreatic necrosis disease (AHPND)) followinglaboratory standardized protocols. Mortality is counted every day forthe next 14 days, and survival shrimps are counted at the end of thisperiod. FIG. 57 provides a proposed timeline for the trial. Tissuesamples of muscle, cephalothorax and haemocytes are taken at thebeginning and end of the feeding period and at the end of the challengeperiod. Tissue is frozen in liquid nitrogen and stored at −80° C. fortranscriptomic analysis if needed. Samples for histopathology analysisare taken during the challenge period as determined by lab pathologist.Parameters to be obtained include final weight, survival rate,histopathological score, and transcriptomic profiles.

Example 10 The Effect of the Composition and/or Combination on Growthand Survival of White Shrimp (Litopenaeus vannamei) Postlarvae Objective

To determine the effect of the composition and/or combination on growthand survival of white shrimp (Litopenaeus vannamei) postlarvae rearingin laboratory conditions. In this trial, the composition and/orcombination was administered as a composition comprising between 15% and40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0%β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase andbetween 1% and 8.0% mannans.

This trial was conducted to determine whether one embodiment of thedisclosed composition and/or combination had an effect on growth or/andsurvival. In the trial postlarvae 12 (PL₁₂) were used because PL₁₀₋₁₂are the most common stage used for stocking into the growout ponds. Thesurvival rate of PL during the first 30 days post-stocking is animportant criteria in shrimp cultivation.

Experimental Animals

Pacific white shrimp postlarvae 9 (PL₉) were used in this study. A totalof 1,000 PL₉ were used and acclimated in fiberglass tanks for 3 days.Later PL₁₂ were used for the trial. Total of 12 500-liter tanks wereused for rearing PL₁₂ with seawater of 20-25 ppt. salinity. Temperaturewas maintained constantly at 29±1° C. by an aquarium heater. PL₁₂ werestocked at a density of 100 PL/m² or 50 shrimp/tank.

The two experimental groups consisted of a control group where theshrimp were fed commercial pelleted feed for white shrimp, and thetreatment group there the shrimp were fed pelleted feed mixed with thecomposition and/or combination at an inclusion rate of 100 mg/kg offeed. Each group comprised six replicates.

Growth and Survival Study

Shrimp were fed four times daily at the satiation rate. Feeding wasadjusted according to shrimp weight throughout the 45 day trial period.Water quality parameters, such as pH, dissolved oxygen (DO), alkalinity,ammonia and nitrate, were maintained at optimal levels for rearingshrimp and analyzed weekly throughout the trial. Survival rate wasrecorded every 10 days. Shrimp were weighed at 30 and 45 days. Feedconversion ratio (FCR) was determined at the end of the trial.

Results

Table 2 and FIG. 59 provide the average survival rates of Pacific whiteshrimp from all groups. At 45 days feeding trial, the highest valueswere seen in the group fed with 200 mg/kg body weight (BW) of thecomposition and/or combination (79.67±7.09%), followed by the group fedwith 100 mg/kg BW of the composition and/or combination (76.68±3.87%)and the lowest values were seen in the control group (75.50±7.42%).

TABLE 2 Average survival rates of Pacific white shrimp at day 0, 10, 20,30, 40 and 45 Average survival rate (%) Treatment Day 0 Day 10 Day 20Day 30 Day 40 Day 45 Control 100 90.85 ± 7.5^(a) 91.83 ± 4.7^(a)   84 ±8.3^(a)   79 ± 4.9^(a)  75.5 ± 7.4^(a) 100 mg/kg 100 97.06 ± 3.9^(a)94.77 ± 7.7^(a) 89.67 ± 8.1^(a) 82.67 ± 7.8^(a) 76.68 ± 3.9^(a) BWComposition and/or combination 200 mg/kg 100 90.85 ± 3.9^(a) 86.27 ±6.4^(a) 86.67 ± 7.5^(a) 80.33 ± 5^(a)   79.67 ± 7.1^(a) BW Compositionand/or combination Data are presented as mean ± standard deviation.Means in the same column with different superscript are significantlydifferent from each other (p < 0.05)

The average body weight of shrimp in all groups were showed in Table 3and FIG. 60. Shrimp in the control group had a lower average body weightof 1.14±0.2 g (at 30 day) and 2.05±0.2 g (at 45 day) compared with thetwo treatment groups (P<0.05) throughout the feeding trial. Shrimp fedwith 100 mg/kg BW of the composition and/or combination had the highestaverage body weight of 1.62±0.1 g (at 30 day) and 2.71±0.3 g (at 45day). While the average body weight of shrimp fed with 200 mg/kg BW ofthe composition and/or combination were 1.48±0.2. (at 30 day) and2.41±0.3 g. (at 45 day).

TABLE 3 Average body weight of Pacific white shrimp at days 30 and 45Average body weight (g) Treatment 30 days 45 days Control 1.14 ± 0.2^(a)2.05 ± 0.2^(a) 100 mg/kg BW Composition 1.62 ± 0.1^(b) 2.71 ± 0.3^(b)and/or combination 200 mg/kg BW Composition 1.48 ± 0.2^(b) 2.41 ±0.3^(b) and/or combination Data are presented as mean ± standarddeviation. Means in the same column with different superscript aresignificantly different from each other (p < 0.05)

The average feed conversion ratio (FCR) is shown in Table 4 and FIG. 61.Shrimp fed with 100 mg/kg BW and 200 mg/kg BW of the composition and/orcombination had a lower feed conversion ratios (FCR) of 1.40±0.1 and1.41±0.04, respectively compared with the control group (1.72±0.10)(p<0.05).

TABLE 4 Feed conversion ratios of Pacific white shrimp after 45 day offeeding with different diets Treatment FCR Control 1.72 ± 0.1^(b)  100mg/kg BW Composition and/or 1.4 ± 0.1^(a) combination 200 mg/kg BWComposition and/or 1.41 ± 0.04^(a) combination Data are presented asmean ± standard deviation. Means in the same column with differentsuperscript are significantly different from each other (p < 0.05)

The water qualities of the groups were not significantly different fromeach other. The water qualities in each tank were controlled within thesuitable range for shrimp cultured throughout the feeding trial (Table5).

TABLE 5 Average water qualities of each treatment group throughout thetrial Treatment Parameter Control 100 mg/kg BW 200 mg/kg BW Temperature(° C.) 28.83 ± 1.1^(a)  29.41 ± 0.1^(a)  28.62 ± 1.2^(a)  Dissolvedoxygen 6.35 ± 0.3^(a)  6.3 ± 0.2^(a) 6.18 ± 0.3^(a) (ppm) pH 8.16 ±0.2^(a) 8.03 ± 0.1^(a) 8.14 ± 0.4^(a) Total alkalinity 150.0 ± 11^(a)  146.67 ± 16.5^(a)  150.0 ± 11^(a  ) (ppm of CaCO₃) Total ammonia- 2.71 ±0.9^(a) 1.50 ± 0.7^(a) 1.59 ± 0.5^(a) nitrogen (ppm) Nitrate-nitrogen0.68 ± 0.6^(a) 0.25 ± 0.2^(a) 0.84 ± 0.5^(a) (ppm) Data are presented asmean ± standard deviation. Means in the same row with differentsuperscript are significantly different from each other (p < 0.05)

Example 11 The Effect of the Composition and/or Combination on Growthand Survival of White Shrimp (Litopenaeus vannamer) Juvenile Challengedwith Pathogenic Bacteria (Vibrio parahaemolyticus) Objective

To determine the effect of the composition and/or combination on growthand survival of white shrimp (Litopenaeus vannamei) juvenile challengedwith pathogenic bacteria (Vibrio harveyi) in laboratory conditions. Inthis trial, the composition and/or combination was administered as acomposition comprising between 15% and 40% silica, between 50% and 81%mineral clay, between 1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3 (4)-endoglucanohydrolase and between 1% and 8.0% mannans.

Early Mortality Syndrome (EMS) cause by V. parahaemolyticus is a commonoccurrence during the growout period between 40-60 days post-stocking.This trial was carried out to determine the composition and/orcombination's effect on growth and survival of shrimp challenged withpathogenic bacteria (V. parahaemolyticus).

Experimental Animals

Juvenile white shrimp weighing 2 g (45 days) from the trial 1 were usedin this study. A total of 30 healthy shrimp were sampled and acclimatedin fiberglass tanks for 7 days before the trial. A total of 12 500-litertanks were used in the trial. Salinity during the acclimation period andsubsequent trial was maintained 20-25 ppt., and temperature wasmaintained constantly at 29±1° C. A virulent strain of V.parahaemolyticus that had been cultured in Tryptic Soy Agar (TSA) with1.5% NaCl (w/v) was added into the tanks at the dose of 104 CFU/mLbefore stocking. This number of bacteria Vibrio is commonly found inpond-reared white shrimp during the culture period.

Two experimental groups with six replicates each were used as in thetrial 1. Five tanks from each group were used for growth and survivalstudy. Another one tank from each group was used for histological study.

Growth and Survival Study

Shrimp were fed four times daily at the satiation rate. Feeding rate wasadjusted according to shrimp weight throughout the 30 days experimentalperiod. Water quality parameters such as pH, dissolved oxygen (DO),alkalinity, ammonia and nitrite were maintained at optimal levels forrearing shrimp and analyzed weekly throughout the experiment. Survivalrate was recorded every 10 days. Shrimp were weighed at days 15 and 30.Mortality and other behavioral responses were recorded throughout the 30days experimental period.

Immune Parameters Study

The immune parameters, comprising phenoloxidase, bacterial activity,phagocytosis activity and total hemocytes, were measured at the end ofthe feeding trial. At least 10 shrimp per treatment were used for theimmunological tests. Blood samples of 0.5 mL per sample were withdrawnfrom the base of the third walking leg of the shrimp by a syringecontaining 1.5 mL anticoagulant (K-199+5% L-cysteine).

1. Total Hemocytes

After collected hemolymph, hemocytes were counted using a hemocytometerand calculated for number blood cells (total hemocytes per cubicmillimeter).

2. Phenoloxidase Activity Assay

After the blood was withdrawn, the hemocytes were washed three timeswith shrimp saline (1,000 rpm at 4° C. for 10 minutes). Hemocyte lysate(HLS) was prepared from hemocytes in a cacodylate buffer at pH 7.4 usinga sonicator at 30 amplitude for 5 seconds followed by centrifuging thesuspension at 10,000 rpm at 4 for 20 minutes.

The supernatant was collected as HLS. Then 200 μl of 0.1% trypsin incacodylate buffer was mixed with the 200 mL HLS followed by 200 μl ofL-dihydroxyphenylalanine (L-DOPA) at 4 mg/mL as a substrate. Enzymeactivity was measured as the absorbance of dopachrome at a wavelength of490 nm, and the protein content in HLS was measured. The phenoloxidaseactivity was calculated as the increasing of optimum density (OD) perminute per mg of protein as:

1 unit of phenoloxidase=ΔOD490/min/mg protein.

3. Bactericidal Activity

Serum was separated from blood of each shrimp sample and diluted by 2.6%NaCl at 1:2, 1:4, 1:8, 1:16 and 1:32. 0.5 mL of each serum dilution and0.5 mL of NaCl as the control were used in the study. 0.5 mL V.parahaemolyticus suspension (prepared from the method as in 2) was putinto each serum dilution and the control. The treatments were incubatedat room temperature for 3 hours before enumerating the number ofbacteria by a spread plate technique, and EC₅₀ values were recorded.

4. Phagocytosis

Two hundred microliters of hemolymph was collected from the base of thethird walking leg of the shrimp and mixed with 800 μL of sterileanticoagulant. Collected shrimp hemocytes were rinsed with shrimp salineand the viable cell number adjusted to 1×10⁶ cells/mL. The cellsuspension (200 μL) was inoculated into a cover slip. After 20 minutes,the cell suspension was removed, and rinsed with shrimp saline threetimes. Heat-killed yeast was added and incubation for 2. hours. Afterthe incubation, the heat-killed yeast was removed, rinsed with shrimpsaline five times, and fixed with 100% methanol. Then, this cover slipwas stained with Giemsa stain and mounted with permount.

Two hundred hemocytes were counted. Phagocytic activity, defined aspercentage phagocytosis, was expressed as

${{Percentage}\mspace{14mu} {phagocytosis}} = {\frac{{phagocytic}\mspace{14mu} {hemocytes}}{{total}\mspace{14mu} {hemocytes}} \times 100}$

Intestinal Bacterial Study and Histological Study

One tank from each group was used for intestinal bacterial andhistological studies. Five shrimp were sampled from each group at 10, 20and 30 days of the trial. The intestine of each shrimp was removed,homogenized and spread on thiosulfate citrate bile salts sucrose agar(TCBS; selective media for Vibrio spp. culture) or nutrient agar (NA;general media for most bacteria culture) by the spreading method, thenincubated at 37° C. for 24 hours. Then all colonies of bacteria werecounted and the CHU/g was calculated.

The head of each shrimp was fixed with Davidson's fixative and processedfor histological study to observe the quality of the hepatopancreas andthe sign of bacterial infection.

Results

The average survival rate of Pacific white shrimp in all groups afterchallenged with Vibrio parahaemolyticus causing Early Mortality Syndromeare shown in Table 6 and FIG. 62.

TABLE 6 Average survival rates of Pacific white shrimp at 0, 15 and 30days Average survival rate (%) Treatment Day 0 Day 15 Day 30 Control100^(a) 81.88 ± 5.2^(a) 74.38 ± 4.7^(a) 100 mg/kg BW 100^(a) 86.96 ±5.6^(a) 75.68 ± 6.1^(a) Composition and/or combination 200 mg/kg BW100^(a) 78.05 ± 7.0^(a) 71.33 ± 4.3^(a) Composition and/or combinationData are presented as mean ± standard deviation. Means in the samecolumn with different superscript are significantly different from eachother (p < 0.05)

The average body weight of shrimp in all groups after 30 days areprovided in Table 7 and FIG. 63. Shrimp in the control group had a loweraverage body weight of 3.31±0.2 g (at 15 day) and 4.67±0.3 g (at 30 day)compared with the two treatment groups (P<0.05) throughout the trial.

TABLE 7 Average body weight of Pacific white shrimp at days 15 and 30Average body weight (g) Treatment 15 days 30 days Control 3.31 ± 0.2^(a)4.67 ± 0.3^(a) 100 mg/kg BW Composition 4.19 ± 0.2^(b) 5.57 ± 0.3^(b)and/or combination 200 mg/kg BW Composition 3.89 ± 0.2^(b) 5.24 ±0.6^(b) and/or combination Data are presented as mean ± standarddeviation. Means in the same column with different superscript aresignificantly different from each other (p < 0.05)

The average feed conversion ratio (FCR) of shrimp in all groups after 30days of the challenge trail are provided in Table 8 and FIG. 64.

TABLE 8 FCR of Pacific white shrimp after 30 day of feeding withdifferent diets Treatment FCR Control 1.95 ± 0.2^(ab) 100 mg/kg BWComposition and/or 1.78 ± 0.05^(a) combination 200 mg/kg BW Compositionand/or 2.01 ± 0.2^(b) combination Data are presented as mean ± standarddeviation. Means in the same column with different superscript aresignificantly different from each other (p < 0.05)

The water qualities in each tank were controlled within the suitablerange for shrimp cultured throughout the challenged trial (Table 9).

TABLE 9 Average water qualities of each treatment group throughout thetrial Treatment Parameter Control 100 mg/kg BW 200 mg/kg BW Temperature(° C.) 28.72 ± 0.6^(a)  28.71 ± 0.6^(a)  28.79 ± 0.6^(a)  Dissolvedoxygen 6.35 ± 0.3^(a)  6.3 ± 0.2^(a)  6.1 ± 0.3^(a) (ppm) pH 8.15 ±0.2^(a) 8.03 ± 0.1^(a) 8.03 ± 0.1^(a) Total alkalinity 150.0 ± 21.8^(a)153.33 ± 19.6^(a)  150.0 ± 11^(a)   (ppm of CaCO₃) Total ammonia- 1.34 ±0.6^(a) 0.56 ± 0.2^(a) 1.02 ± 0.9^(a) nitrogen (ppm) Nitrate-nitrogen 0.09 ± 0.05^(a)   0.07 ± 0.03^(a)  0.1 ± 0.03^(a) (ppm) Data arepresented as mean ± standard deviation. Means in the same row withdifferent superscript are significantly different from each other (p <0.05)

The average total number of bacteria in the intestine of shrimp areshown in Table 10 and FIG. 65. And the average total number of Vibrioparahaemolyticus in the intestine of shrimp is shown in Table 11 andFIG. 66. And immunological results are provided by Tables 12-16 andFIGS. 67-70.

TABLE 10 Average number of bacteria (10⁵ CFU/g) in the intestine ofPacific white shrimp after a challenge with V. parahaemolyticus (10⁴CFU/g) for 30 days Total number of bacteria (10⁵ CFU/g) in the intestineTreatment 10 days 20 days 30 days Control 7.69 ± 3.8^(a) 59.83 ± 9.2^(a)13.88 ± 4^(a)   100 mg/kg BW 3.01 ± 1.4^(a) 50.32 ± 7.9^(a) 11.80 ±8.2^(a) Composition and/or combination 200 mg/kg BW 4.38 ± 3.9^(a) 53.54± 8.6^(a) 13.06 ± 3.1^(a) Composition and/or combination Data arepresented as mean ± standard deviation. Means in the same column withdifferent superscript are significantly different from each other (p <0.05)

TABLE 11 Average number of Vibrio spp. (10⁵ CFU/g) in the intestine ofPacific white shrimp after a challenge with V. parahaemolyticus (10⁴CFU/g) for 30 days Total number of Vibrio spp. (10⁵ CFU/g) in theintestine Treatment 10 days 20 days 30 days Control 0.20 ± 0.01^(a) 2.29± 0.4^(a) 3.70 ± 5^(a)   100 mg/kg BW 0.08 ± 0.06^(a) 2.04 ± 0.1^(a)3.03 ± 0.5^(a) Composition and/or combination 200 mg/kg BW 0.18 ±0.02^(a) 2.16 ± 0.4^(a) 3.17 ± 0.5^(a) Composition and/or combinationData are presented as mean ± standard deviation. Means in the samecolumn with different superscript are significantly different from eachother (p < 0.05)

TABLE 12 Total hemocyte count (10⁷ cells/mL) of Pacific white shrimpafter challenge with V. parahaemolyticus (10⁴ CFU/g) for 30 daysTreatment THC (10⁶ cells/mL) Control  11.3 ± 7.7^(a) 100 mg/kg BWComposition and/or 29.5 ± 14^(a) combination 200 mg/kg BW Compositionand/or 31.8 ± 10^(a) combination Data are presented as mean ± standarddeviation. Means in the same column with different superscript aresignificantly different from each other (p < 0.05)

TABLE 13 The phagocytosis activity (%) of Pacific white shrimp afterchallenge with V. parahaemolyticus (10⁴ CFU/g) for 30 days TreatmentPhagocytosis (%) Control 21.00 ± 8.2^(a) 100 mg/kg BW Composition and/or23.17 ± 7.7^(a) combination 200 mg/kg BW Composition and/or 21.00 ±4.6^(a) combination Data are presented as mean ± standard deviation.Means in the same column with different superscript are significantlydifferent from each other (p < 0.05)

TABLE 14 The phenoloxidase activity (units/min/mg protein) of Pacificwhite shrimp after challenge with V. parahaemolyticus (10⁴ CFU/g) for 30days Phenoloxidase Treatment (units/min/mg protein) Control 159.33 ±16.5^(a) 100 mg/kg BW Composition and/or 172.11 ± 10^(a)   combination200 mg/kg BW Composition and/or 164.04 ± 5.8^(a)  combination Data arepresented as mean ± standard deviation. Means in the same column withdifferent superscript are significantly different from each other (p <0.05)

TABLE 15 The superoxide dismutase activity (SOD) units/mL) of Pacificwhite shrimp after challenge with V. parahaemolyticus (10⁴ CFU/g) for 30days Treatment SOD (SOD units/mL) Control 11.22 ± 1.8^(a) 100 mg/kg BWComposition and/or 11.05 ± 2.4^(a) combination 200 mg/kg BW Compositionand/or 12.84 ± 1^(a)   combination Data are presented as mean ± standarddeviation. Means in the same column with different superscript aresignificantly different from each other (p < 0.05)

TABLE 16 The bactericidal activity of Pacific white shrimp afterchallenge with V. parahaemolyticus (10⁴ CFU/g) for 30 days TreatmentBactericidal activity Control 1:8 100 mg/kg BW Composition and/or 1:8combination 200 mg/kg BW Composition and/or 1:8 combination

Histopathological study of the hepatopancreas of the studies shrimprevealed signs of cell necrosis in the hepatopancreas, caused by toxinfrom Vibrio parahaemolyticus infections. The percentage of necrosis cellin the hepatopancreas of the shrimp from each group was similar at about25-30% (FIGS. 71-76).

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A composition and/or combination formulated foradministration to an aquatic species, comprising a feedstuff for anaquatic species, an adhesive agent, and a composition comprising:glucan, silica, mineral clay, and mannans; yucca and quillaja; orglucan, silica, mineral clay, mannans, yucca and quillaja; thecomposition and/or combination having a particle size of from greaterthan 0 mm to 3 mm.
 2. The composition and/or combination of claim 1,comprising: a first composition comprising glucan, silica, mineral clay,mannans, and an endoglucanohydrolase, the first composition adhered tothe feedstuff for an aquatic species by a first adhesive agent toproduce a first solid material having a first particle size of fromgreater than 0 mm to 3 mm; a second composition comprising yucca andquillaja, the second composition adhered to the feedstuff for an aquaticspecies by a second adhesive agent to produce a second solid materialhaving a second particle size of from greater than 0 mm to 3 mm; or acomposition or combination comprising the first and second compositions.3. The composition and/or combination of claim 1, wherein thecomposition and/or combination comprises from greater than zero to atleast 10% adhesive agent by weight.
 4. The composition and/orcombination of claim 1, wherein the adhesive agent is an oil or a syrup.5. The composition and/or combination of claim 4, wherein the adhesiveagent is selected from soy oil, linseed oil, tung oil, dehydrated castoroil, canola oil, olive oil, palm oil, cottonseed oil, Naskole oil,molasses, sugar syrup, sorghum, honey or a combination thereof;
 6. Thecomposition and/or combination of claim 4, wherein the adhesive agentcomprises at least 2 wt % soy oil.
 7. The composition and/or combinationof claim 2, wherein the composition and/or combination comprises thefirst composition.
 8. The composition and/or combination of claim 7,wherein the first composition comprises between 15% and 40% silica,between 50% and 81% mineral clay, between 1.0% and 5.0% β-glucans, andbetween 1% and 8.0% mannans.
 9. The composition and/or combination ofclaim 7, wherein the first composition further comprises yucca,quillaja, or yucca and quillaja.
 10. The composition and/or combinationof claim 7, wherein the first composition further comprises at least oneBacillus species.
 11. The composition and/or combination of claim 7,wherein the first composition consists essentially of between 15% and40% silica, between 50% and 81% mineral clay, between 1.0% and 5.0%β-glucans, between 0.05% and 3.0% β-1,3 (4)-endoglucanohydrolase,between 1% and 8.0% mannans, and/or at least 2% of the first adhesiveagent.
 12. The composition and/or combination of claim 2, wherein thecomposition and/or combination comprises the second composition.
 13. Thecomposition and/or combination of claim 1, wherein the feedstuff is afeed ration, a mineral supplement, a protein supplement, a premix, orany combination thereof.
 14. The composition and/or combination of claim1, wherein the composition and/or combination is a composition.
 15. Thecomposition and/or combination of claim 1, wherein the compositionand/or combination is a composition having a particle size of fromgreater than 0 mm to 3 mm and comprising the feedstuff, at least 2% soyoil, and from 15% to 40% silica, from 50% to 81% mineral clay, from 1.0%to 5.0% β-glucans, from 0.05% to 3.0% β-1,3 (4)-endoglucanohydrolase,and from 1% to 8.0% mannans in amounts relative to each other.
 16. Amethod, comprising administering the composition and/or combination ofclaim 1 to an aquatic animal.
 17. The method of claim 16, wherein thecomposition and/or combination is administered to the animal to promotegrowth, to reduce its feed conversion ratio, or both.
 18. The method ofclaim 16, wherein administering the composition and/or combinationenhances a feed conversion ratio of the aquatic animal by from 0.5% to20%, compared to a feed conversion ratio of an aquatic animal that isnot administered the composition and/or combination.
 19. The method ofclaim 16, wherein the composition and/or combination is administered tothe animal to prevent, delay or ameliorate at least one deleterioussymptom or sign.
 20. The method of claim 16, wherein the compositionand/or combination is administered to the animal in an amount from 50mg/kg of body weight per day to 250 mg/kg of body weight per day. 21.The method of claim 16, wherein the composition and/or combination isused as a feedstuff.
 22. The method of claim 16, wherein the aquaticanimal is selected from a fish, crustacean, or mollusk.
 23. The methodof claim 22, wherein: the fish is selected from salmon, trout, tilapia,sea bream, carp, cod, halibut, snapper, herring, catfish, flounder,hake, smelt, anchovy, lingcod, moi, perch, orange roughy, bass, tuna,mahi mahi, mackerel, eel, barracuda, marlin, Atlantic ocean perch, Nileperch, Arctic char, haddock, hoki, Alaskan Pollock, turbot, freshwaterdrum, walleye, skate, sturgeon, Dover sole, common sole, wolfish,sablefish, American shad, John Dory, grouper, monkfish, pompano, lakewhitefish, tilefish, wahoo, cusk, bowfin, kingklip, opah, mako shark,swordfish, cobia, croaker, or hybrids thereof; the crustacean isselected from shrimp, crab, lobster, crayfish, krill, copepods,barnacles, or hybrids thereof; or the mollusk is selected from squid,octopus, clam, oyster, mussel, abalone, conch, rock snail, whelk,cockle, or hybrids thereof.
 24. The method of claim 22, wherein: thefish is selected from Nile tilapia, blue tilapia, Mozambique tilapia,tilapiine cichlids, sheepshead, scup, yellowfin bream, gilt-head bream,Saucereye porgies, red sea bream, common carp, Asian carp, Indian carp,black carp, grass carp, silver carp, bighead carp, pink salmon, chumsalmon, sockeye salmon, coho salmon, Atlantic salmon, chinook salmon,masu salmon, rainbow trout, Adriatic trout, Bonneville cutthroat trout,brook trout, steelhead trout, Atlantic northeast cod, Atlantic northwestcod, Pacific cod, Pacific halibut, Atlantic halibut, red snapper,bluefish, Atlantic herring, Pacific herring, channel catfish, walkingcatfish, shark catfish, Corydoras, basa, banjo catfish, talking catfish,long-whiskered catfish, armoured suckermouth catfish, blue catfish, orhybrids thereof; the crustacean is selected from Chinese white shrimp,pink shrimp, black tiger shrimp, freshwater shrimp, gulf shrimp, Pacificwhite shrimp, whiteleg shrimp, giant tiger shrimp, rock shrimp, Akiamapaste shrimp, Southern rough shrimp, fleshy prawn, banana prawn,Northern prawn, blue crab, peekytoe crab, spanner crab, Jonah crab, snowcrab, king crab, stone crab, Dungeness crab, soft-shell crab, Cromercrab, American lobster, spiny lobster, squat lobster, goose barnacle,picoroco barnacle, or hybrids thereof; or the mollusk is selected fromcommon squid, Patagonian squid, longfin inshore squid, neon flyingsquid, Argentine shortfin squid, Humboldt squid, Japanese flying squid,Wellington squid, common octopus, hard clam, soft-shell clam, oceanquahog, surf clam, Asari, Hamaguri, Vongola, Cozza, Tellina, Pacificoyster, rock oyster, European flat oyster, Portuguese oyster, bluemussel, freshwater mussel, green-lipped mussel, Asian green mussel,Mediterranean mussel, Baltic mussel, or hybrids thereof.